Opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of Gram positive bacteria

ABSTRACT

The present invention encompasses monoclonal antibodies that bind to lipoteichoic acid (LTA) of Gram positive bacteria. The antibodies also bind to whole bacteria and enhance phagocytosis and killing of the bacteria in vitro. The invention also provides antibodies having human sequences (chimeric, humanized and human antibodies). The invention also sets forth the variable regions of three antibodies within the invention and presents the striking homology between them.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/097,055, filed Jun. 15, 1998 (Attorney DocketNo. 7787.0041), and is further based on and claims the benefit of U.S.Provisional Application S. No. 60/343,503, filed Dec. 21, 2001 (AttorneyDocket No. 7787.6008). The entire disclosure of this provisionalapplication is relied upon and incorporated by reference herein. Thisapplication also relates to U.S. Pat. No. 5,571,511, U.S. Pat. No.5,955,074, and U.S. patent application Ser. No. 09/097,055, filed Jun.15, 1998, all of which are specifically incorporated herein byreference.

DESCRIPTION OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention in the fields of immunology and infectiousdiseases relates to antibodies that are specific for Gram positivebacteria, particularly to bacteria that bear lipoteichoic acids on theirsurfaces. The invention includes monoclonal and chimeric antibodies, aswell as fragments, regions and derivatives thereof. This inventionfurther relates to sequences of the variable region that enhance theantibody's opsonic activity. The antibodies of the invention may be usedfor diagnostic, prophylactic and therapeutic applications.

[0004] 2. Background of the Invention

[0005] The search for agents to combat bacterial infections has beenlong and arduous. The development of antibiotics has brought us from thetime when sepsis associated with amputation was associated with a 50percent mortality rate. Today's challenge, however, is the increasingdevelopment of bacteria that are resistant to antibiotics, such asmembers of the genera Staphylococcus.

[0006] Staphylococci are particularly worrisome because they commonlycolonize humans and animals and are an important cause of humanmorbidity and mortality. Because of their prevalence on the skin andmucosal linings, staphylococci are ideally situated to produce bothlocalized and systemic infections. Of the staphylococci, both S. aureus,a coagulase positive bacteria, and S. epidermidis, a coagulase negativespecies, are the most problematic. In fact, S. aureus is the mostvirulent Staphylococcus, producing severe and often fatal disease inboth normal and immunocompromised hosts. S. epidermidis has become oneof the major causes of nosocomial (hospital acquired) infection inpatients with impaired immune responses or those whose treatmentsinvolve the placement of foreign objects into the body, such as patientswho receive continuous ambulatory peritoneal dialysis and patientsreceiving parenteral nutrition through central venous catheters (25).Indeed, S. epidermidis is now recognized as a common cause of neonatalnosocomial sepsis, and infections frequently occur in premature infantsthat have received parenteral nutrition. Moreover, in recent years, theinvolvement of S. epidermidis in neonatal infection has increaseddramatically. Indeed, for every 10 babies diagnosed with bacterialsepsis seven or more days after birth (indicative of post-partumbacterial exposure), six of those are infected with S. epidermidis.Untreated, Staphylococcus infections in newborns can result in multipleorgan failure and death in two to three days. Antibiotics are onlypartially effective and, unfortunately, the rise in multiply drugresistant strains of Staphylococcus renders antibiotic treatments lessand less effective.

[0007] The problems of antibiotic resistance are so significant thatthey have reached the lay press. See, e.g., The Washington Post “Microbein Hospital Infections Show Resistance to Antibiotics,” May 29, 1997;The Washington Times, “Deadly bacteria outwits antibiotics,” May 29,1997. And this concern is borne out by the scientific literature. See L.Garrett, The Coming Plague, “The Revenge of the Germs or Just KeepInventing New Drugs” Ch. 13, pgs. 411-456, Farrar, Straus and Giroux,N.Y., Eds. (1994). In one study, the majority of staphylococci isolatedfrom blood cultures of septic infants were resistant to multipleantibiotics (10). Another study describes methicillin-resistant S.aureus (31). There is no doubt that the emergence of antibioticresistance among clinical isolates is making treatment difficult (18).

[0008] The other possible route of treatment is the administration ofantibodies. Antibodies protect against bacterial attack by recognizingand binding to antigens on the bacteria to thereby facilitate theremoval or “clearance” of the bacteria by a process called phagocytosis,wherein phagocytic cells (predominantly neutrophils and macrophages)identify, engulf, and subsequently destroy the invading bacteria.However, bacteria have developed mechanisms to avoid phagocytosis, suchas the production of a “capsule” to which phagocytes cannot adhere orthe production of toxins that actually poison the encroachingphagocytes. Antibodies overcome these defenses by, for example, bindingto the toxins to thereby neutralize them. More significantly, antibodiesmay themselves bind to the capsule to coat it, in a process calledopsonization, to make the bacteria extremely attractive to phagocytesand to enhance their rate of clearance from the bloodstream.

[0009] Confounding the use of administered antibodies, however, areconflicting reports in the literature. For example, the immunizationstudies of Fattom et al. demonstrated that opsonization of S.epidermidis was related to the specific capsule type, as with S. aureusand other encapsulated Gram positive bacteria such as Streptococcuspneumonia (6). In another study, Timmerman et al. identified a surfaceprotein of S. epidermidis that induced opsonic monoclonal antibodies(39). Timmerman et al. also identified other monoclonal antibodies thatbound to non-homologous S. epidermidis strains, but only the monoclonalantibody produced to the homologous strain was opsonic, thusopsonization was enhanced only to the homologous strain but not toheterologous strains. Accordingly, based on the studies of Fattom etal., and Timmerman et al., and others in the field (and in contrast toour own studies as set forth in U.S. Pat. Nos. 5,571,511 and 5,955,074),one would not expect that an antibody that is broadly reactive tomultiple strains of S. epidermidis and to S. aureus would have opsonicactivity against each strain. This is particularly true for antibodiesthat bind to both coagulase positive and coagulase negativestaphylococci.

[0010] Further exacerbating the problem, the role of the common surfaceantigens on staphylococci has been unclear. For example, whilelipoteichoic acid and teichoic acid make up the majority of the cellwall of S. aureus, there was no prior appreciation that antibodies tolipoteichoic acid and teichoic acid could be protective. Indeed,anti-teichoic acid antibodies have been often used as controls. Forexample, Fattom et al. examined the opsonic activity of antibodiesinduced against a type-specific capsular polysaccharide of S.epidermidis, using as controls antibodies induced against teichoic acidsand against S. hominus. While type-specific antibodies were highlyopsonic, anti-teichoic acid antibodies were not functionally differentfrom the anti-S. hominus antibodies (6).

[0011] Similarly, in Kojima et al., the authors assessed the protectiveeffects of antibody to capsular polysaccharide/adhesion againstcatheter-related bacteremia due to coagulase negative staphylococci andspecifically used a strain of S. epidermidis that expresses teichoicacid as a control ((16); see page 436, Materials and Methods, leftcolumn, first paragraph; right column, third paragraph). In a laterstudy, Takeda et al. (38), the authors reached a more explicitconclusion against the utility of anti-techoic antibodies:

[0012] Immunization protocols designed to elicit antibody to techoicacid but not to PS/A afforded no protection against bacteremia orendocarditis (38).

[0013] Thus, the role of antibodies in the protection against infectionsby Gram positive bacteria, particularly Staphylococci such as S. aureusand S. epidermidis, has not been clear, and there is a need in the artfor monoclonal antibodies to both protect against such bacterialinfection and to help elucidate the role of such antibodies against suchinfection. There is also a need in the art for sequence analysis of suchantibodies so that antibodies of enhanced binding and opsonic activitycan be identified and/or produced.

SUMMARY OF THE INVENTION

[0014] The present invention encompasses broadly reactive, opsonic, andprotective monoclonal and chimeric antibodies that bind to lipoteichoicacid (LTA) of Gram positive bacteria. The antibodies also bind to wholebacteria and enhance phagocytosis and killing of the bacteria in vitroand enhance protection from lethal infection in vivo. The presentinvention further encompasses opsonic antibodies to LTA that share ahigh degree of sequence homology. The present invention also encompassesantibodies having variable regions derived from two or more differentanti-LTA antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 provides a schematic representation of lipoteichoic acid(LTA) in the Gram positive bacterial cell wall.

[0016]FIG. 2 depicts antibody regions, including the heavy chainconstant region (C_(H)), the heavy chain variable region (V_(H)), thelight chain constant region (C_(L)), and the light chain variable region(V_(L)). The complementarity determining regions (CDRs) within thevariable regions are shown as black bars.

[0017]FIG. 3 shows the cDNA cloning strategy for the heavy and lightchain variable regions of A120.

[0018]FIG. 4 shows the oligonucleotide primers used to amplify thevariable region fragments. (SEQ ID NOs: 1-9 and 18)

[0019]FIG. 5 shows the amino acid sequence (SEQ ID NO: 10) and thepolynucleotide sequence (SEQ ID NO: 11) of the A120 light chain variableregion.

[0020]FIG. 6 shows the amino acid sequence (SEQID NO: 12) and thepolynucleotide sequence (SEQ ID NO: 13) of the A120 heavy chain variableregion.

[0021]FIG. 7 depicts the pJSB23-1 plasmid that expresses the A120 heavychain.

[0022]FIG. 8 depicts the pJSB24 plasmid that expresses the A120 lightchain.

[0023]FIG. 9 shows an alignment of (A) the A110 lightchain variableregion cDNA (SEQ ID NO: 14), the A120 light chain variable region cDNA(SEQ ID NO: 11) and the 391.4 light chain variable region cDNA (SEQ IDNO: 19) and (B) the A110 heavy chain variable region cDNA (SEQ ID NO:15), the A120 heavy chain variable region cDNA (SEQ ID NO: 13) and the391.4 heavy chain variable region cDNA (SEQ ID NO: 20). The nucleotidesthat differ between any two sequences are boxed.

[0024]FIG. 10A shows an alignment of the A110 light chain variableregion polypeptide sequence (SEQ ID NO: 16), the A120 light chainvariable region polypeptide sequence (SEQ ID NO: 10) and the 391.4 lightchain variable region polypeptide sequence (SEQ ID NO: 21). FIG. 10Bshows an alignment of the A110 heavy chain variable region polypeptidesequence (SEQ ID NO: 17), the A120 heavy chain variable regionpolypeptide sequence (SEQ ID NO: 12) and the 391.4 heavy chain variableregion polypeptide sequence (SEQ ID NO: 22). The complementaritydetermining regions (CDRs) are underlined and the amino acids thatdiffer between any two sequences are boxed.

[0025]FIG. 11 depicts the pJRS354 bi-cistronic plasmid that expressesthe A110 heavy chain and light chain variable regions.

[0026]FIG. 12 depicts the pJSB25-3 bi-cistronic plasmid that expressesthe A110 heavy chain variable region and the A120 light chain variableregion.

[0027]FIG. 13 depicts the pJSB26 bi-cistronic plasmid that expresses theA120 heavy chain and light chain variable regions.

[0028]FIG. 14 depicts the pJSB27 bi-cistronic plasmid that expresses theA120 heavy chain variable region and the A110 light chain variableregion.

[0029]FIG. 15 provides the results of the chimeric antibody productionELISA. All antibodies shown are human/mouse chimeras. A110 contains boththe heavy and light chain variable regions from A110. A120 contains boththe heavy and light chain variable regions from A120. A120a contains theheavy chain variable region from A110 and the light chain variableregion from A120. A120b contains the heavy chain variable region fromA120 and the light chain variable region from A110.

[0030]FIG. 16 provides the results of the experiment to determinechimeric antibody binding to the S. aureus LTA. The antibodies used arethe same as in FIG. 15.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Definitions

[0032] The term “antibody”, as used herein, includes full-lengthantibodies and portions thereof. A full-length antibody has one pair or,more commonly, two pairs of polypeptide chains, each pair comprising alight and a heavy chain. Each heavy or light chain is divided into tworegions, the variable region (which confers antigen recognition andbinding) and the constant region (associated with localization andcellular interactions). Thus, a full-length antibody commonly containstwo heavy chain constant regions (H_(C) or C_(H)), two heavy chainvariable regions (H_(V) or V_(H)), two light chain constant regions(L_(C) or C_(L)), and two light chain variable regions (L_(V) or V_(L))(FIG. 2). The light chains or chain, may be either a lambda or a kappachain. Thus, in an embodiment of the invention, the antibodies includeat least one heavy chain variable region and one light chain variableregion, such that the antibody binds antigen.

[0033] Another aspect of the invention involves the variable region thatcomprises alternating complementarity determining regions, or CDRs, andframework regions, or FRs. The CDRs are the sequences within thevariable region that generally confer antigen specificity.

[0034] The invention also encompasses portions of antibodies thatcomprise sufficient variable region sequence to confer antigen binding.Portions of antibodies include, but are not limited to Fab, Fab′,F(ab′)₂, Fv, SFv, scFv (single-chain Fv), whether produced byproteolytic cleavage of intact antibodies, such as papain or pepsincleavage, or by recombinant methods, in which the cDNAs for the intactheavy and light chains are manipulated to produce fragments of the heavyand light chains, either separately, or as part of the same polypeptide.

[0035] MAbs within the scope of the invention include sequencescorresponding to human antibodies, animal antibodies, and combinationsthereof. The term “chimeric antibody,” as used herein, includesantibodies that have variable regions derived from an animal antibody,such as a rat or mouse antibody, fused to another molecule, for example,the constant domains derived from a human antibody. One type of chimericantibodies, “humanized antibodies”, have had the variable regionsaltered (through mutagenesis or CDR grafting) to match (as much aspossible) the known sequence of human variable regions. CDR graftinginvolves grafting the CDRs from an antibody with desired specificityonto the FRs of a human antibody, thereby replacing much of thenon-human sequence with human sequence. Humanized antibodies, therefore,more closely match (in amino acid sequence) the sequence of known humanantibodies. By humanizing mouse monoclonal antibodies, the severity ofthe human anti-mouse antibody, or HAMA, response is diminished. Theinvention further includes fully human antibodies which would avoid, asmuch a possible, the HAMA response.

[0036] Modified antibodies include, for example, the proteins orpeptides encoded by truncated or modified antibody-encoding genes. Suchproteins or peptides may function similarly to the antibodies of theinvention. Other modifications, such as the addition of other sequencesthat may enhance the effector function, which includes the ability toblock or alleviate nasal colonization by staphylococci, are also withinthe present invention. Such modifications include, for example, theaddition of amino acids to the antibody's amino acid sequence, deletionof amino acids in the antibody's amino acid sequence, substitution ofone or more amino acids in the antibody amino acid sequence withalternate amino acids, isotype switching, and class switching.

[0037] In certain embodiments, an antibody may be modified in its Fcregion to prevent binding to bacterial proteins. The Fc region normallyprovides binding sites for neutrophils, macrophages, other accessorycells, complement components, and, receptors of the immune system. Asthe antibodies bind to bacteria and opsonize them, accessory cellsrecognize the coated bacteria and respond to infection. When a bacterialprotein binds to the Fc region near the places where accessory cellsbind, the normal function of these cells is inhibited. For example,Protein A, a bacterial protein found in the cell membrane of S. aureus,binds to the Fc region of IgG near accessory cell binding sites. Indoing so, Protein A inhibits the function of these accessory cells, thusinterfering with clearance of the bacterium. To circumvent thisinterference with the antibacterial immune response, the Fc portion ofthe antibody of the invention may be modified to prevent nonspecificbinding of Protein A while retaining binding to accessory cells (15).

[0038] In light of these various forms, the antibodies of the inventioninclude clones of full length antibodies, antibody portions, chimericantibodies, humanized antibodies, fully human antibodies, and modifiedantibodies. Collectively, these will be referred to as “MAbs” ormonoclonal antibodies unless otherwise indicated.

[0039] The term “epitope”, as used herein, refers to a region, orregions, of LTA that is bound by an antibody to LTA. The regions thatare bound may or may not represent a contiguous portion of the molecule.

[0040] The term “antigen”, as used herein, refers to a polypeptidesequence, a non-proteinaceous molecule, or any molecule that can berecognized by the immune system. An antigen may be a full-sizedstaphylococcal protein or molecule, or a fragment thereof, wherein thefragment is either produced from a recombinant cDNA encoding less thanthe full-length protein or derived from the full-sized molecule orprotein. Such fragments may be produced via enzymatic processing, suchas proteolysis. An antigen may also be a polypeptide sequence thatencompasses an epitope of a staphylococcal protein, wherein the epitopemay not be contiguous with the linear polypeptide sequence of theprotein. The DNA sequence encoding an antigen may be identified,isolated, cloned, and transferred to a prokaryotic or eukaryotic cellfor expression by procedures well-known in the art (25).

[0041] An antigen, or epitope thereof, may be 100% identical to a regionof the staphylococcal molecule or protein amino acid sequence, or it maybe at least 95% identical, or at least 90% identical, or at least 85%identical. An antigen may also have less than 95%, 90% or 85% identitywith the staphylococcal molecule or protein amino acid sequence,provided that it still be able to elicit antibodies the bind to a nativestaphylococcal molecule or protein. The percent identity of a peptideantigen can be determined, for example, by comparing the sequence of thetarget antigen or epitope to the analogous portion of staphylococcalsequence using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The GAP programutilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443,1970), as revised by Smith and Waterman (Adv. Appl. Math2:482,1981), and is applicable to determining the percent identity ofprotein or nucleotide sequences referenced herein. The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745,1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358,1979; (2) a penalty of 3.0 for each gapand an additional 0.10 penalty for each symbol in each gap; and (3) nopenalty for end gaps.

[0042] Alternatively, for simple comparisons over short regions up to 10or 20 units, or regions of relatively high homology, for example betweenantibody sequences, the percent identity over a defined region ofpeptide or nucleotide sequence may by determined by dividing the numberof matching amino acids or nucleotides by the total length of thealigned sequences, multiplied by 100%. Where an insertion or gap of one,two, or three amino acids occurs in a MAb chain, for example in orabutting a CDR, the insertion or gap is counted as single amino acidmismatch.

[0043] Antigens may be surface antigens and/or virulence antigens and/oradherance antigens. Surface antigens are antigens that are accessible toan antibody when the antigen is in the configuration of the whole intactbacterium, i.e., the antigen is not inside the cell cytoplasm. Virulenceantigens are antigens that are involved in the pathogenic process,causing disease in a host. Virulence antigens include, for example, LTA,peptidoglycan, toxins, fimbria, flagella, and adherence antigens.Adherence antigens mediate the ability of a staphylococcal bacterium toadhere to an epithelial surface, such as the epithelial surface of theanterior nares. An antigen may be a non-proteinaceous component ofstaphylococci such as a carbohydrate or lipid. For example,peptidoglycan and lipoteichoic acid are two non-proteinaceous antigensfound in the cell wall of staphylococci. Antigens may comprise orinclude fragments of non-proteinaceous molecules as long as they elicitan immune response.

[0044] As used herein, antigens include molecules that can elicit anantibody response to LTA. An antigen may be LTA itself, or a fragment orportion thereof. An antigen may also be an unrelated molecule, which,through some structural similarity, is able to elicit antibodies thatbind to LTA. Binding to LTA may thus be assessed by binding to suchpeptide epitope mimics, as described, for example, in U.S. Ser. No.09/893,615, incorporated herein by reference. In certain embodiments ofthe invention, an antigen elicits antibodies that bind to LTA on thesurface of bacteria.

[0045] As specifically used herein, an antigen is any molecule that canspecifically bind to an antibody, including antibodies specific for LTA.Antigens of the invention thus include antigens that bind to any ofmonoclonal antibodies MAb-391.4, M110, M120, A110, A120, A120a, andA120b, described herein.

[0046] An antibody is said to specifically bind to an antigen, epitope,or protein, if the antibody gives a signal by an assay such as an ELISAassay that is at least two fold, at least three fold, at least fivefold, or at least ten fold greater than the background signal, i.e., atleast two fold, at least three fold, at least five fold, or at least tenfold greater than the signal ascribed to non-specific binding. Anantibody is said to specifically bind to a bacterium if the antibodygives a signal by MeOH-fixed bacteria ELISA or live bacteria ELISA, orother assay, that is at least 1.5 fold, 2 fold, or 3 fold greater thanthe background signal.

[0047] “Enhanced phagocytosis”, as used herein, means an increase inphagocytosis over a background level as assayed by the methods in thisapplication, or another comparable assay. The level deemed valuable maywell vary depending on the specific circumstances of the infection,including the type of bacteria and the severity of the infection. Forexample, for enhanced phagocytic activity, in one embodiment, anenhanced response is equal to or greater than 75% over backgroundphagocytosis. In another embodiment, an enhanced response is equal to orgreater than 80% or 85% over background phagocytosis. In anotherembodiment, an enhanced response is equal to or greater than 90% or 95%over background phagocytosis. Enhanced phagocytosis may also be equal toor greater than 50%, 55%, 60%, 65%, or 70% over background phagocytosis.In another embodiment, enhanced phagocytosis comprises a statisticallysignificant increase in phagocytic activity as compared to backgroundphagocytosis or phagocytosis with a non-specific or non-opsonic controlantibody.

[0048] The specific determination or identification of a “statisticallysignificant” result will depend on the exact statistical test used. Oneof ordinary skill in the art can readily recognize a statisticallysignificant result in the context of any statistical test employed, asdetermined by the parameters of the test itself. Examples of thesewell-known statistical tests include, but are not limited to, X² Test(Chi-Squared Test), Students t Test, F Test, M test, Fisher Exact Text,Binomial Exact Test, Poisson Exact Test one way or two way repeatedmeasures analysis of variance, and calculation of correlation efficient(Pearson and Spearman).

[0049] A MAb has “opsonic activity” if it can bind to an antigen topromote attachment of the antigen to the phagocyte and thereby enhancephagocytosis. As used herein, opsonic activity may also be assessed byassays that measure neutrophil mediated opsonophagocytotic bactericidalactivity.

[0050] The MAb's of the invention are useful for the treatment ofsystemic and local staphylococcal infections. As used herein,“treatment” encompasses any reduction, amelioration, or “alleviation” ofexisting infection as well as “blocking” or prophylaxis against futureinfection. In this respect, treatment with a MAb of the invention issaid to “alleviate” staphylococcal nasal colonization if it is able todecrease the number of colonies in the nares of a mammal when the MAb isadministered before, concurrently with, or after exposure tostaphylococci, whether that exposure results from the intentionalinstillation of staphylococcus or from general exposure. For instance,in the nasal colonization animal model described below, a MAb orcollection of MAbs is considered to alleviate colonization if the extentof colonization, or the number of bacterial colonies that can be grownfrom a sample of nasal tissue, is decreased after administering the MAbor collection of MAbs. A MAb or collection of MAbs alleviatescolonization in the nasal colonization assays described herein when itreduces the number of colonies by at least 50%, at least 60%, at least75%, at least 80%, or at least 90%. 100% alleviation may also bereferred to as eradication.

[0051] A MAb is said to “block” staphylococcal colonization if it isable to prevent the nasal colonization of a human or non-human mammalwhen it is administered prior to, or concurrently with, exposure tostaphylococci, whether by intentional instillation or otherwise into thenares. A MAb blocks colonization, as in the nasal colonization assaydescribed herein, if no staphylococcal colonies can be grown from asample of nasal tissue taken from a mammal treated with the MAb of theinvention for an extended period such as 12 hours or longer or 24 hoursor longer compared to control mammals. A MAb also blocks colonization inthe nasal colonization assay described herein if it causes a reductionin the number of animals that are colonized relative to control animals.For instance, a MAb is considered to block colonization if the number ofanimals that are colonized after administering the material and theGram-positive bacteria is reduced by at least 25%, at least 50%, and atleast 75%, relative to control animals or if no colonies can be grownfrom a sample taken from a treated individual for an extended periodsuch as 12 hours or 24 hours or longer.

[0052] In a clinical setting, the presence or absence of nasalcolonization in a human patient is determined by culturing nasal swabson an appropriate bacterial medium. These cultures are scored for thepresence or absence of staphylococcal colonies. In this type ofqualitative assay system, it may be difficult to distinguish betweenblocking and alleviation of staphylococcal colonization. Thus, for thepurposes of qualitative assays, such as nasal swabs, a MAb “blocks”colonization if it prevents future colonization in human patients whoshow no signs of prior colonization for an extended period of 12 or 24hours or longer. A MAb “alleviates” colonization if it causes adiscernable decrease in the number of positive cultures taken from ahuman patient who is already positive for staphylococci before the MAbsof the invention are administered.

[0053] A vaccine is considered to confer a protective immune response ifit stimulates the production of opsonic antibodies to gram-positivebacteria. Production of opsonic antibodies may be measured by thepresence of such antibodies in the serum of a test subject that has beenadministered the vaccine, relative to a control that has not receivedthe vaccine. The presence of opsonic antibodies in the serum may bemeasured by the activity assays described herein, or by other equivalentassays. If an opsonophagocytic bactericidal assay is used, then killingby the test serum of at least 50% more bacteria, 75% more bacteria, andat least 100% more bacteria, relative to the control serum, isconsidered to be enhanced immunity.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides murine antibodies, includingmonoclonal antibodies, and chimeric, humanized and fully humanantibodies, fragments, derivatives, and regions thereof, which bind tolipoteichoic acid (LTA) of Gram positive staphylococci. Gram positivebacteria, unlike Gram negative bacteria, take up the Gram stain as aresult of a difference in the structure of the cell wall. The cell wallsof Gram negative bacteria are made up of a unique outer membrane of twoopposing phospholipid-protein leaflets, with an ordinary phospholipid inthe inner leaflet but the extremely toxic lipopolysaccharide in theouter leaflet. The cell walls of Gram positive bacteria seem muchsimpler in comparison, containing two major components, peptidoglycanand teichoic acids plus additional carbohydrates and proteins dependingon the species.

[0055] Moreover, because the basis of the binding to Gram positivebacteria is the presence of LTA and because LTA is a major component ofthe cell walls of Gram positive bacteria and is highly conserved, theantibodies of the claimed invention are broadly reactive against Grampositive bacteria. This broad reactivity permits the antibodies of theinvention to block the binding of Gram positive bacteria to epithelialcells, such as human epithelial cells (50-54). Finally, these antibodiesexhibit broad opsonic activity and consequently enhance phagocytosis andkilling of Gram positive bacteria. Accordingly, the invention providesbroadly reactive, opsonic, and protective antibodies for the diagnosis,prevention, and/or treatment of bacterial infections caused by Grampositive bacteria.

[0056] Among the Gram positive Staphylococci against which theantibodies of the invention are directed are S. aureus (a coagulasepositive bacteria) and S. epidermidis (a coagulase negative bacteria).

[0057] Three of the monoclonal antibodies of the invention (M110, M120,and MAb-391.4) bind stronglyto LTA. M110 and M120 also exhibit highopsonic activity for S. epidermidis, while MAb-391.4 is also opsonic forS. epidermidis, but less so. M120 is also highly opsonic against S.aureus. M110 was derived from mice immunized with whole S. epidermidisstrain Hay as described in detail in U.S. patent application Ser. No.09/097,055, filed Jun. 15, 1998, incorporated by reference. In screeningfor hybridomas, the antibodies of one clone (hybridoma line 96-105CE11IF6, which produces antibody M110) were found to bind very strongly toGram positive bacteria such as strain Hay, all three serotypes of S.epidermidis, S. hemolyticus, S. hominus, and two serotypes of S. aureus,but not to the Gram negative control, Haemophilus influenza (see U.S.patent application Ser. No. 09/097,055).

[0058] M120 was derived from mice immunized with conjugates of S. aureusLTA. The antibodies of one clone (00-107GG12 ID12, which producesantibody M120) were found to bind strongly to LTA, and were opsonic forS. aureus type 5 and S. epidermidis strain Hay.

[0059] MAb-391.4 is from QED Biosciences, and was derived from miceimmunized with whole UV-killed S. aureus.

[0060] The variable regions of M110, M120, and 391.4 were sequenced andcompared, revealing a surprising 88% identity (203/230) at the aminoacid level. Further, the level of identity was found to be 96% (220/230)between the antibodies that are highly opsonic for S. epidermidis, M110and M120. We believe that this level of homology between threemonoclonal antibodies that were raised in three different mice, usingthree different antigen preparations from two different types ofbacteria, is unprecedented. To understand how unexpected this findingis, one need only consider how vast and diverse is the collection ofantibodies in the immune system.

[0061] The immune system is made up of a large number of B cells, eachbearing antibodies of a different specificity, but only about 1 in10,000 to 1 in 1,000,000 B cells is specific for a particular antigen.When a foreign antigen, such as is found on the surface of a bacteria,enters the blood stream, the appropriate B cell recognizes that antigenand then enters a lymph node where it undergoes rapid division toproduce many progeny bearing the identical specificity. However, therapidly dividing B cells also undergo somatic hypermutation. Somatichypermutation results in about half of the B cells acquiring mutationsin their rearranged heavy and light chain genes, with mutation occurringpreferentially in complementarity determining regions (CDRs) of thevariable regions. Mutated B cells that retain their ability to bindantigen continue to secrete antibody, while those that no longer bindantigen undergo apoptosis. As the antigen is cleared from the host, onlyB cells that have very high antigen affinity survive in a process calledaffinity maturation. The surviving activated B cells differentiate intoplasma cells, which are short-lived and secrete antibody, and memory Bcells, which are long-lived lymphocytes bearing membrane-bound antibodythat can be rapidly stimulated when the antigen is re-introduced.

[0062] The processes of somatic hypermutation and affinity maturationresult in progeny B cells that are of higher affinity and haveimmunoglobulins of different amino acid sequence than the originalactivated B cell. Therefore, a single B cell that is activated by aforeign antigen can produce many progeny of differing affinity andimmunoglobulin amino acid sequence.

[0063] Because of these processes, it is generally believed that twoanimals immunized with the same antigen will produce vastly differentantibody repertoires. Nickerson and colleagues demonstrated this conceptwhen they showed that a mouse monoclonal antibody and a human monoclonalantibody that showed nearly identical binding to the same blood group Aantigen shared only 15% and 37% identity in their heavy and light chainCDRs (55). X-ray crystallography studies of two antibodies that bothbind to hemagglutinin of influenza virus, reveal that, although theyshare only 56% sequence identity, they both bind with similar affinitiesand in the same orientation to the same epitope (56).

[0064] It has been postulated that the immune system has evolved toprovide a maximum range of antigen specificities and redundancy, ratherthan to bind to specific antigens (55). It follows, therefore, thatantibodies derived from the same mouse may be of high specificity, butlow homology, because any number of progenitor B cells may be specificfor the immunized antigen. Amplification and somatic mutation of thoseprogenitors may, however, result in groups of antibodies that are ofhigher homology within the group, although they are of very low homologybetween groups. Antibodies raised against the same immunogen in two ormore different mice will necessarily be even less homologous, becausethey do not share progenitor B cells.

[0065] Three specific antibodies of the present invention, M110, M120,and MAb-391.4, were not only raised in different mice, but withdifferent immunogens: A110 was raised to whole S. epidermidis, M120 wasraised to purified and conjugated S. aureus LTA, and MAb-391.4 wasraised to whole UV-killed S. aureus. Yet, though these antibodies wereraised against different immunogen preparations in different mice, theyshare 88% identity at the amino acid level in both the heavy and lightchain variable regions. This high degree of homology suggests that LTAcontains a highly antigenic, and highly conserved, epitope which isbound by the three antibodies in a very similar manner. This epitope andmode of binding may be responsible for the high opsonic activity of themonoclonal antibodies.

[0066] MAb-391.4 and human/mouse chimeric antibodies of M110 and M120,designated A110 and A120, respectively, were tested for opsonicactivity. MAb-391.4, A110, and A120 each demonstrated a high level ofopsonic activity against S. epidermidis strain Hay. (See also See U.S.patent application Ser. No. 09/097,055).

[0067] MAb A110 is currently being manufactured under GMP conditions inpreparation for clinical trials. Additional disclosure regarding the MAbA110 is provided in U.S. Provisional Application S. No. 60/341,806, andin related application Methods for Blocking or AlleviatingStaphylococcal Nasal Colonizaton by Intranasal Application of MonoclonalAntibodies, filed concurrently herewith, both of which are expresslyincorporated by reference.

[0068] Thus, one aspect of the invention relates to antibodies that bindto the LTA of Gram positive bacteria, including both coagulase negative(S. epidermidis) and coagulase positive (S. aureus) bacteria, and thatenhance the opsonization of such bacteria. These anti-LTA antibodiesinclude monoclonal antibodies, such as M110, M120, and MAb-391.4,chimeric monoclonal antibodies A110, A120, A120a, and A120b, and othermonoclonal antibodies including, chimeric, humanized, fully humanantibodies, antibody fragments, and modified antibodies.

[0069] In a one aspect of the invention, as noted above, the antibody isa chimeric mouse/human antibody made up of regions from the anti-LTAantibodies of the invention together with regions of human antibodies.Chimeric or other monoclonal antibodies are advantageous in that theyavoid the development of anti-murine antibodies. In at least one study,patients administered murine anti-TNF (tumor necrosis factor) monoclonalantibodies developed anti-murine antibody responses to the administeredantibody (5). This type of immune response to the treatment regimen,commonly referred to as the human anti-mouse antibody response, or theHAMA response, decreases the effectiveness of the treatment and may evenrender the treatment completely ineffective. Humanized or chimerichuman/mouse monoclonal antibodies have been shown to significantlydecrease the HAMA response and to increase the therapeutic effectiveness(19).

[0070] Thus, in one aspect of the invention, a chimeric heavy chain cancomprise the antigen binding region of the heavy chain variable regionof the anti-LTA antibody of the invention linked to at least a portionof a human heavy chain IgG, IgA, IgM, or IgD constant region. Thishumanized or chimeric heavy chain may be combined with a chimeric lightchain that comprises the antigen binding region of the light chainvariable region of the anti-LTA antibody linked to at least a portion ofthe human light chain kappa or lambda constant region. Exemplaryembodiments include, but are not limited to, an antibody having a mouseheavy chain variable region fused to a human IgG₁ constant region, and amouse light chain variable region fused to a human kappa light chainconstant region.

[0071] The chimeric antibodies and other MAb's of the invention may bemonovalent, divalent, or polyvalent immunoglobulins. For example, amonovalent chimeric antibody is a dimer (HL) formed by a chimeric Hchain associated through disulfide bridges with a chimeric L chain, asnoted above. A divalent chimeric antibody is a tetramer (H₂ L₂) formedby two HL dimers associated through at least one disulfide bridge. Apolyvalent or multivalent chimeric antibody may be based on anaggregation of chains, with or without a carrier or scaffold.

[0072] The MAbs of the invention include antibodies that contain heavyand light chain variable regions derived from two different antibodies.In one embodiment, the heavy and light chain variable regions arederived from two antibodies that bind to the same molecule, e.g. LTA.Exemplary embodiments include A120a, which is a human/mouse chimericantibody that has a heavy chain variable region from A110 and a lightchain variable region from A120; and A120b, which is a human/mousechimeric antibody that has a heavy chain variable region from A120 and alight chain variable region from A110. Additional exemplary embodimentsinclude antibodies that comprise a heavy chain variable region fromMAb-391.4, and a light chain variable region from either of A110 orA120, and antibodies that comprise a light chain variable region fromMAb-391.4, and a heavy chain variable region from either of A110 orA120.

[0073] In yet another aspect, the invention is a collection of opsonicmonoclonal antibodies that bind to LTA and that exhibit a high degree ofhomology in the variable regions at either the amino acid or nucleicacid level, or both. In one embodiment, this collection comprises one ormore of M110,M120, their human/mouse chimeric counterparts, A110, A120,and MAb-391.4. In one aspect, the amino acid sequences of the variableregions are at least 75% identical, at least 80% identical, at least 85%identical, at least 88% identical, at least 90% identical, or at least95% identical as defined above.

[0074] In addition to the antibodies, the present invention alsoencompasses the DNA sequences of the genes coding for the antibodies(see, e.g., FIGS. 5, 6, and 9; SEQ ID NOs: 11, 13-15, 19, and 20) aswell as the polypeptides encoded by the DNA (see, e.g., FIGS. 5, 6, and10; SEQ ID NOs: 10, 12, 16, 17, 21, and 22). Those figures provide thevariable regions of the heavy and light chains of A110, A120, andMAb-391.4, including the complementarity determining regions (CDRs), thehypervariable amino acid sequences within antibody variable regions thatusually interact with the antigen. As noted above, the DNA and aminoacid sequence homology between A110 and A120 is striking. There is a 94%homology (216/229) at the amino acid level and a 96% homology (662/687)at the DNA level between the antibodies. This suggests that theseantibodies share a sequence and structural similarity.

[0075] The invention includes peptide sequences for, and DNA sequencesencoding, full-length antibodies and portions thereof, as well as CDRsand FRs relating to these MAbs. The invention further includes DNA andpeptide sequences that are homologous to these sequences. In oneembodiment, these homologous DNAs and peptide sequences are about 70%identical, although other embodiments include sequences that about 75%,80%, 85%, 88%, 90%, and 95% or more identical. As indicated above,determining levels of identity for both the DNA and peptide sequence iswell within the routine skill of those in the art.

[0076] As shown in FIG. 10A, alignment of the A110, A120, and 391.4light chain variable regions (Seq. ID Nos. 16, 10, and 21, respectively)shows identical amino acids in 95 of 106 amino acids, or more than 89%identity overall. Within the region spanning the CDRs (amino acids 24 to96 of the light chain variable regions) the percent identity is about93% (68 our of 73 amino acids). It is predicted that light chainvariable regions with a somewhat lower overall identity would still formMAbs that specifically bind LTA, and are therefore within the scope ofthe invention. The CDRs themselves show at least 88% identity, inparticular, CDR1 (amino acids 24-33), CDR2 (amino acids 49-55), and CDR3(amino acids 88-73), show 9/10, 7/7, and 8/9 identical amino acids.Likewise, the framework regions (FRs) surrounding the CDRs are alsohighly conserved: amino acids 1-23 of SEQ ID Nos. 16, 10, and 21 showgreater than 86% identity (20/23 matching amino acids); amino acids34-38 show about 93% identity (14/15); amino acids 56-87 show about 93%identity (68/73); and amino acids 97-106 show 70% identity.

[0077] Similarly, in FIG. 10B, alignment of the A110, A120, and 391.4heavy chain variable regions (Seq. ID Nos. 17, 12, and 22, respectively)also shows a high degree of sequence identity. Counting single aminoacid gaps and insertions as single-point mis-matches, Seq. ID Nos. 17,12, and 22 show 86% identity overall (108/125 identical amino acids). Itis predicted that heavy chain variable regions with a somewhat loweroverall identity would still form MAbs that specifically bind LTA, andare therefore within the scope of the invention. The degree of identityis particularly high in the FR region preceding CDR1 through the FRregion preceding CDR3, in particular, the 96 base region from amino acid16 to 101 of Seq. ID Nos. 17, 12, and 22 shows 8 mismatches orapproximately 91% identity. CDR1, itself, shows 90% identity over 10amino acids (amino acids 26-35), and CDR2 (amino acids 50-69) showsabout 89% identity over 19 amino acids. The framework regionssurrounding the CDRs are also highly conserved. Amino acids 1-25 of SEQID Nos. 17, 12, and 22 show 92% identity (23/25 matching amino acids);amino acids 36-49 show 100% identity over 14 amino acids; the FR regionbetween CDR2 and CDR3 (amino acids 70 to about 101) shows about 87%identity (over 31-32 amino acids); and amino acids 115-125 show 90%identity.

[0078] Thus, in one aspect, the invention encompasses polypeptides(including regions of larger polypeptides, such as MAbs) that 1) exhibithigh sequence homology to Seq. ID Nos. 10, 12, 16, 17, 21, or 22, ordefined regions thereof, and 2) are capable of functioning as all orpart of the variable region of a MAb that specifically binds LTA. In oneembodiment, such polypeptides comprise, or are at least 70%, 75%, 77%80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, 95% identical to, any ofSeq. ID Nos. 10, 12, 16, 17, 21, or 22. Conversely, polypeptides withinthe scope of the invention may be less than 100%, 99%, 95%, 90%, 80% orless identical to Seq. ID Nos. 10, 12, 16, 17, 21, or 22 provided thatthey are capable of functioning as all or part of the variable region ofa MAb that specifically binds to LTA.

[0079] In another embodiment, polypeptides within the scope of theinvention comprise, or are at least 70%, 75%, 77%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 93%, 95% identical to, amino acids 24 to 96 of anyof Seq. ID Nos. 10, 16, or 21. In another embodiment, such polypeptidescomprise, or are at least 70%, 75%, 77%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 93%, 95% identical to, 1) amino acids 24-33, 49-55, and 88-73of Seq. ID Nos. 10, 16, or 21, or 2) amino acids 26-35 or 50-69 of Seq.ID Nos. 12, 17, or 22; and are capable of functioning as a CDR, orportion thereof, in a MAb that specifically binds to LTA. In anotherembodiment, such polypeptides comprise, or are at least 70%, 75%, 77%,80%, 81%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, 95% identicalto, 1) amino acids 1-23, 34-38, 56-87, and 97-106 of Seq. ID Nos. 10,12, 16, 17, 21, or 22, or 2) amino acids 1-25, 36-49, 70-101, or 115-125of Seq. ID Nos. 12, 17, or 22; and are capable of functioning as aframework region, or portion thereof, in a MAb that specifically bindsto LTA.

[0080] The invention further comprises collections of a multiplicity ofany of the above sequences capable of functioning as all or part of thevariable region of a MAb that specifically binds to LTA, as part of alarger polypeptide, MAb, collection of MAbs or aggregation of MAbs; andthe use thereof in prophylaxis, treatment, and for the production ofpharmaceutical compounds or medicaments. The invention further comprisesany non-naturally occurring RNA, DNA, or vector thereof, encoding any ofthe above sequences capable of functioning as all or part of thevariable region of a MAb that specifically binds to LTA, as well asplasmids, viruses, bacteria, yeast, microorganisms, cell lines,transgenic plants or animals harboring or expressing such nucleic acids.Thus, the invention contemplates production systems for Mabs, lightchains, heavy chains, and portions thereof, comprising 1) a cell(including bacteria, yeast, microorganisms, eukaryotic cell lines,transgenic plant or animal) in connection with 2) at least onerecombinant nucleic acid capable of directing the expression of any ofthe Mabs or related polypeptides of the invention.

[0081] The invention thus further comprises a general method ofidentifying highly antigenic and highly conserved epitopes by raisingantibodies against different immunogen preparations in different mice,sequencing the variable regions of the antibodies, comparing thevariable regions, and identifying antibodies that share a high degree ofhomology in the variable regions.

[0082] The DNA sequences of the invention can be identified, isolated,cloned, and transferred to a prokaryotic or eukaryotic cell forexpression by procedures well-known in the art. Such procedures aregenerally described in Molecular Cloning: A Laboratory Manual, as wellas Current Protocols in Molecular Biology (44, 45), which areincorporated by reference. Guidance relating more specifically to themanipulation of sequences of the invention may be found in AntibodyEngineering, and Antibodies: A Laboratory Manual (64, 65), both of whichare incorporated by reference in their entirety. In certain embodiments,a CDR can be grafted onto any human antibody framework region usingtechniques standard in the art, in such a manner that the CDR maintainsthe same binding specificity as in the intact antibody. As noted asabove, an antibody that has its CDRs grafted onto a human frameworkregion is said to be “humanized”. Humanized, and fully human antibodiesgenerally also include human constant regions, thus maximizing thepercentage of the antibody that is human-derived, and potentiallyminimizing the HAMA response.

[0083] In addition, the DNA and peptide sequences of the antibodies ofthe invention, including both monoclonal and chimeric antibodies,humanized and fully human antibodies, may form the basis of antibody“derivatives,” which include, for example, the proteins or peptidesencoded by truncated or modified genes. Such proteins or peptides mayfunction similarly to the antibodies of the invention. Othermodifications, such as the addition of other sequences that may enhancethe effector function, which includes phagocytosis and/or killing of thebacteria are also within the present invention.

[0084] The present invention also discloses a pharmaceutical compositioncomprising the antibodies, whether monoclonal or chimeric, humanized, orfully human, together with a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the invention may alternatively comprisethe isolated antigen, epitope, or portions thereof, together with apharmaceutically acceptable carrier.

[0085] Pharmaceutically acceptable carriers can be sterile liquids, suchas water, oils, including petroleum oil, animal oil, vegetable oil,peanut oil, soybean oil, mineral oil, sesame oil, and the like. Salinesolutions, aqueous dextrose, and glycerol solutions can also be employedas liquid carriers, particularly for injectable solutions. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, 18th Edition (13), which is herein incorporated by reference.

[0086] Additionally, the invention may be practiced with variousdelivery vehicles and/or carriers. Such vehicles may increase thehalf-life of the MAbs in storage and upon administration including, butnot limited to, application to skin, wounds, eyes, lungs, or mucusmembranes of the nasal or gastrointestinal tract, or upon inhalation orinstillation into the nares. These carriers comprise natural polymers,semi-synthetic polymers, synthetic polymers, lipososmes, and semi-soliddosage forms (21, 29, 33, 35, 36, 46). Natural polymers include, forexample, proteins and polysaccharides. Semi-synthetic polymers aremodified natural polymers such as chitosan, which is the deacetylatedform of the natural polysaccharide, chitin. Synthetic polymers include,for example, polyphosphoesters, polyethylene glycol, poly (lactic acid),polystyrene sulfonate, and poly (lactide coglycolide). Semi-solid dosageforms include, for example, dendrimers, creams, ointments, gels, andlotions. These carriers can also be used to microencapsulate the MAbs orbe covalently linked to the MAbs.

[0087] Finally, the present invention provides methods for treating apatient infected with, or suspected of being infected with, aGram-positive bacteria such as a staphylococcal organism. The methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising the anti-LTA immunoglobulin(whether monoclonal, chimeric, humanized, or fully human, includingfragments, regions, and derivatives thereof) and a pharmaceuticallyacceptable carrier. A patient can be any human or non-human mammal inneed of prophylaxis or other treatment. Representative patients includeany mammal subject to S. aureus or other staphylococcal or Gram-positiveinfection or carriage, including humans and non-human animals such asmice, rats, rabbits, dogs, cats, pigs, sheep, goats, horses, primates,ruminants including beef and milk cattle, buffalo, camels, as well asfur-bearing animals, herd animals, laboratory, zoo, and farm animals,kenneled and stabled animals, domestic pets, and veterinary animals.

[0088] A therapeutically effective amount is an amount reasonablybelieved to provide some measure of relief, assistance, prophylaxis, orpreventative effect in the treatment of the infection. A therapeuticallyeffective amount may be an amount believed to be sufficient to block abacterial infection. Similarly, a therapeutically effective amount maybe an amount believed to be sufficient to alleviate a bacterialinfection. Such therapy as above or as described below may be primary orsupplemental to additional treatment, such as antibiotic therapy, for astaphylococcal infection, an infection caused by a different agent, oran unrelated disease. Indeed, combination therapy with other antibodiesis expressly contemplated within the invention.

[0089] A further embodiment of the present invention is a method ofpreventing such infections, comprising administering a prophylacticallyeffective amount of a pharmaceutical composition comprising the anti-LTAantibody (whether monoclonal, chimeric, humanized, or fully human) and apharmaceutically acceptable carrier.

[0090] A prophylactically effective amount is an amount reasonablybelieved to provide some measure of prevention of infection by Grampositive bacteria. Such therapy as above or as described below may beprimary or supplemental to additional treatment, such as antibiotictherapy, for a staphylococcal infection, an infection caused by adifferent agent, or an unrelated disease. Indeed, combination therapywith other antibodies is expressly contemplated within the invention.

[0091] The antibodies and the pharmaceutical compositions of theinvention may be administered by intravenous, intraperitoneal,intracorporeal injection, intraarticular, intraventricular, intrathecal,intramuscular or subcutaneous injection, or intranasally, dermally,intradermally, intravaginally, orally, or by any other effective methodof administration. The composition may also be given locally, such as byinjection to the particular area infected, either intramuscularly orsubcutaneously. Administration can comprise administering thepharmaceutical composition by swabbing, immersing, soaking, or wipingdirectly to a patient. The treatment can also be applied to objects tobe placed within a patient, such as dwelling catheters, cardiac valves,cerebrospinal fluid shunts, joint prostheses, other implants into thebody, or any other objects, instruments, or appliances at risk ofbecoming infected with a Gram positive bacteria, or at risk ofintroducing such an infection into a patient.

[0092] As a particularly valuable corollary of treatment with thecompositions of the invention (pharmaceutical compositions comprisinganti-LTA antibodies, whether, monoclonal, chimeric, humanized or fullyhuman) may be the reduction in cytokine release that results from theintroduction of the LTA of a Gram positive bacteria (49). As is nowrecognized in the art, LTA induces cytokines, including, for example,tumor necrosis factor alpha, interleukin 6, and interferon gamma (see,e.g., (37)). Accordingly, the compositions of the invention may enhanceprotection at three levels: (1) by binding to LTA on the bacteria andthereby blocking the initial binding to epithelial cells and preventingsubsequent invasion of the bacteria; (2) by binding to LTA on bacteriaand thereby enhancing opsonization of the bacteria and clearance of thebacteria from tissues and/or blood; and/or (3) by binding to LTA andpartially or fully blocking cytokine release and modulating theinflammatory responses to prevent shock and tissue destruction.

[0093] Having generally described the invention, it is clear that theinvention overcomes some of the potentially serious problems describedin the Background section regarding the development of antibioticresistant Gram positive bacteria. As set forth above, Staphylococci andStreptococci (such as S. faecalis) have become increasingly resistant toantibiotics and, with the recent spread of vancomycin resistant strains,antibiotic therapy may become totally ineffective.

[0094] Particular aspects of the invention are now presented in the formof the following Materials and Methods, as well as the specificExamples. Of course, these are included only for purposes ofillustration and are not intended to be limiting of the presentinvention.

Materials and Methods

[0095] Bacteria

[0096]S. aureus, type 5, is deposited at the ATCC under Accession No.49521.

[0097]S. epidermidis, strain Hay, was deposited at the ATCC on Dec. 19,1990 under Accession No. 55133.

[0098] Hybridoma

[0099] Hybridoma 96-105CE11 IF6 (M110) was deposited at the ATCC on Jun.13, 1997, under Accession No. HB-12368.

[0100] Hybridoma 00-107GG12 ID12 (M120) was deposited at the ATCC onAug. 16, 2001, under Accession No. PTA-3644.

[0101] Hybridoma 391.4 was deposited at the ATCC on Dec. 18, 2001, underAccession No. PTA-3932.

[0102] Isotype Determination Assay

[0103] Isotype was determined using a mouse immunoglobulin isotype kitobtained from Zymed Laboratories (Cat. No. 90-6550).

[0104] Binding Assays

[0105] In the binding assays of the invention, immunoglobulin isincubated with a preparation of whole cell staphylococci or with apreparation of bacterial cell wall components such as LTA or PepG. Thebinding assay may be an agglutination assay, a coagulation assay, acalorimetric assay, a fluorescent binding assay, or any other suitablebinding assay that is known in the art. A particularly suitable assay iseither an enzyme-linked immunosorbent assay (ELISA) or aradio-immunoassay (RIA). Binding is detected directly and can also bedetected indirectly by using competitive or noncompetitive bindingprocedures known in the art.

[0106] The whole cell staphylococcus preparation, LTA preparation, PepGpreparation, or a combination of those preparations, may be fixed usingstandard techniques to a suitable solid support, including, but notlimited to, a plate, a well, a bead, a micro-bead, a paddle, apropeller, or a stick. Solid supports may be comprised of, for example,glass or plastic. In certain embodiments of the invention, the solidsupport is a microtiter plate.

[0107] Generally, a binding assay requires the following steps. First,the fixed preparation is incubated with an immunoglobulin source. In oneembodiment of the assay, the immunoglobulin source is, for example,tissue culture supernatant or a biological sample such as ascites,plasma, serum, whole blood, or body tissue. In another embodiment, theimmunoglobulin may be further isolated or purified from its source bymeans known in the art, and the purified or isolated immunoglobulin issubsequently used in the assay. The amount of binding is determined bycomparing the binding in a test sample to the binding in a negativecontrol. A negative control is defined as any sample that does notcontain antigen-specific immunoglobulin. In the binding assay, apositive binding reaction results when the amount of binding observedfor the test sample is greater than the amount of binding for a negativecontrol. Positive binding may be determined from a singlepositive/negative binding reaction or from the average of a series ofbinding reactions. The series of binding reactions may include samplescontaining a measured amount of immunoglobulin that specifically bindsto the fixed antigen, thereby creating a standard curve. This standardcurve may be used to quantitate the amount of antigen-specificimmunoglobulin in an unknown sample.

[0108] In an alternate embodiment of the assay, antibodies are fixed toa solid support and an unknown immunoglobulin sample is characterized byits ability to bind a bacterial preparation. The other aspects of theassays discussed above apply where appropriate.

[0109] The specific binding assays used in the Examples are set forthbelow:

[0110] Live Bacteria ELISA (LBE): The LBE assay was performed to measurethe ability of antibodies to bind to live bacteria. Various types ofbacteria may be used in this assay, including S. aureus type 5, type5-USU, type 8, S. epidermidis strain Hay, and S. hemolyticus. Bacteriafrom an overnight plate culture were transferred to 35 mis of TrypticSoy Broth (TSB) and grown with gentle shaking for 1.5-2.0 hours at 37°C. The bacteria were then pelleted by centrifugation at 1800-2000×g for15 minutes at room temperature. The supernatant was removed and thebacteria were resuspended in 35-45 mis of phosphate buffered salinecontaining 0.1% bovine serum albumin (PBS/BSA). The bacteria were againpelleted by centrifugation, the supernatant discarded and the bacteriaresuspended in PBS/BSA to a percent transmittance (%T) of 65%-70% at 650nm. From this suspension the bacteria were further diluted 15-fold insterile 0.9% sodium chloride (Sigma cat. no. S8776, or equivalent), and100 μl of this suspension was added to replicate wells of aflat-bottomed, sterile 96-well plate.

[0111] Each antibody to be tested was diluted to the desiredconcentration in PBS/BSA containing 0.05% Tween-20 and horse radishperoxidase-conjugated Protein A (Protein A-HRP, Zymed Laboratories) at a1:8000 dilution (PBS/BSA/Tween/Prot A-HRP). The Protein A-HRP wasallowed to bind to the antibodies for 30-60 minutes at room temperaturebefore use, thereby generating an antibody-Protein A-HRP complex tominimize the potential non-specific binding of the antibodies to theProtein A found on the surface of S. aureus. Generally, severaldilutions of test antibody were used in each assay. From each antibodydilution, 50 μl of the antibody-Protein A-HRP complex was added toreplicate wells and the mixture of bacteria and antibody-Protein A-HRPcomplex was incubated at 37° C. for 30-60 minutes with gentle rotation(50-75 rpm) on an orbital shaker.

[0112] Following the incubation, the bacteria were pelleted in the plateby centrifugation at 1800-2000×g for 10-15 minutes at room temperature.The supernatant was carefully removed from the wells and 200 μl ofPBS/BSA containing 0.05% Tween-20 (PBS/BSA/Tween) was added to all wellsto dilute unbound reagents. The bacteria were again pelleted bycentrifugation and the supernatant was removed. Two hundred μl ofPBS/BSA/Tween was again added to all wells and the bacteria were againpelleted by centrifugation as described above. The supernatant wasremoved and 100 μl of TMB substrate (BioFx, Inc. cat. no. TMBW-0100-01,or equivalent) was added to each well and the hydrolysis of thesubstrate was allowed to proceed for 15 minutes at room temperature. Thereactions were stopped by adding 100 μl of TMB stop reagent (450 nm StopReagent; BioFx, Inc. catalog no. STPR-0100-01, or equivalent). Theabsorbance of each well was determined using a microplate reader fittedwith a 450 nm filter.

[0113] In this assay, the intensity of the color development wasdirectly proportional to the binding of the antibodies to the bacteria.Control wells contained bacteria and Protein A-HRP without antibody.

[0114] Immunoassay on Methanol-Fixed Bacteria: Heat-killed bacteria weresuspended in sterile 0.9% sodium chloride (Sigma cat. no. S8776, orequivalent) at a % transmittance (%T) of 70-75% at 650 nm. Tenmilliliters the bacterial suspension was diluted 15-fold in sterile 0.9%sodium chloride and then pelleted by centrifugation at 1800×g for 15minutes at 10-15° C. The supernatant was discarded and the pellet wasresuspended in 15 milliliters of methanol (MeOH). One hundredmicroliters of the bacteria-MeOH suspension was distributed into eachwell of Nunc Maxisorp Stripwells (Nunc catalog no. 469949). The MeOH wasallowed to evaporate, fixing the bacteria to the plastic wells. Thebacteria-coated stripwells were stored in plastic bags in the dark atroom temperature and used within 2 months of preparation.

[0115] For evaluation of antibodies, the bacteria-coated plates werewashed four times with phosphate buffered saline containing 0.05%Tween-20 (PBS-T) as follows. Approximately 250 microliters of PBS-T wasadded to each well. The buffer was removed by flicking the plate overthe sink and the remaining buffer removed by inverting the plate andtapping it on absorbent paper. The antibody was diluted in PBS-T andthen added to the wells. Supernatants, ascites, or purified antibodieswere tested at the dilutions indicated in the Examples. Control wellsreceived PBS-T alone. After addition of the antibody, the wells wereincubated at room temperature for 30-60 minutes in a draft-freeenvironment. The wells were again washed four times with PBS-T.Ninety-five microliters of detection antibody was then added to eachwell. The detection antibody was one of the following: rabbit anti-mouseIgG₃, rabbit anti-mouse IgM, or goat anti-human IgG (gamma-specific),all conjugated to horse radish peroxidase (HRP) and diluted 1:6000 inPBS-T (Zymed catalog numbers 61-0420, 61-6820 and 62-8420,respectively).

[0116] Following another 30-60 minute incubation at room temperature,the wells were washed four times with PBS-T and each well received 100pi of TMB substrate solution (BioFx #TMBW-0100-01). Plates wereincubated in the dark at room temperature for 15 minutes and the bindingreactions were stopped by the addition of 100 μl of TMB stop solution(BioFx #STPR-0100-01). The absorbance of each well was measured at 450nm using a Molecular Devices Vmax plate reader.

[0117] Immunoassay with Protein A: In order to evaluate the binding ofthe MAbs to S. aureus, the immunoassay procedure was modified formethanol-fixed bacteria, described above. Because S. aureus expressesProtein A on its surface, and Protein A binds strongly to the constantregion of the heavy chains of gamma-globulins, it is possible that falsepositive results may be obtained from non-specific binding of theantibodies to Protein A. To overcome this difficulty, the immunoassaywells were coated with bacteria as described above, but prior to theaddition of the antibodies to the bacteria-coated wells, the MAbs wereincubated with a solution of recombinant Protein A conjugated to HRP(Zymed Laboratories Cat. No. 10-1123), diluted 1:10,000 in PBS-T. Thebinding reaction was allowed to proceed for 30 minutes at roomtemperature. The wells were washed four times with PBS-T and 100 μl ofthe solution of each Protein A-HRP-MAb combination was added to thewells. The presence of the Protein A-HRP from the pretreatment minimizedthe binding of the MAbs to the Protein A on the S. aureus. Furthermore,the binding of the Protein A-HRP to the constant region of the heavychain did not interfere with the antibody binding site on the MAbs,thereby allowing evaluation of the MAbs on S. aureus and other bacteria.

[0118] The Protein A-HRP-MAb solutions were allowed to bind in thecoated wells for 30-60 minutes at room temperature. The wells were thenwashed with PBS-T and TMB substrate solution was added and the assaycompleted as described above.

[0119] Immunoassay on LTA: The binding of the MAbs to LTA was measuredby immunoassay on wells coated with S. aureus LTA (Sigma Cat. No. 2515).One hundred microliters of a 1 μg/ml LTA solution in PBS was distributedinto replicate Nunc Maxisorp Stripwells and incubated overnight at roomtemperature. The unbound material was removed from the wells by washingfour times with PBS-T. Antibody, diluted in PBS-T, was then added to thewells and the assay continued as described above for the Immunoassay onMethanol-Fixed Bacteria.

[0120] For immunoassays on PepG, Nunc Maxisorp Stripwell plates werecoated with 100 μl of a 5-10 μg/ml solution of PepG (gift of S. Foster)in 0.1 M carbonate buffer (pH 9.2-9.6) overnight at room temperature.Unbound antigen was removed from the plate by washing four times withPBS-T. Antibody, diluted in PBS-T, was then added to the wells and theassay continued as described above for the Immunoassay on Methanol-FixedBacteria.

[0121] Activity Assays

[0122] Antibodies that bind to an antigen may not necessarily enhanceopsonization or enhance protection from infection. Therefore, anopsonization assay was used to determine the functional activities ofthe antibodies.

[0123] An opsonization assay can be a calorimetric assay, achemiluminescent assay, a fluorescent or radiolabel uptake assay, acell-mediated bactericidal assay, or any other appropriate assay knownin the art which measures the opsonic potential of a substance andthereby identifies reactive immunoglobulin. In an opsonization assay, aninfectious agent, a eukaryotic cell, and the opsonizing substance to betested, or an opsonizing substance plus a purported opsonizing enhancingsubstance, are incubated together.

[0124] In certain embodiments, the opsonization assay is a cell-mediatedbactericidal assay. In this in vitro assay, an infectious agent such asa bacterium, a phagocytic cell, and an opsonizing substance such asimmunoglobulin, are incubated together. Any eukaryotic cell Withphagocytic or binding ability may be used in a cell-mediatedbactericidal assay. In certain embodiments, phagocytic cells aremacrophages, monocytes, neutrophils, or any combination of these cells.Complement proteins may be included to promote opsonization by both theclassical and alternate pathways.

[0125] The opsonic ability of an antibody is determined by the amount ornumber of infectious agents remaining after incubation. The fewer thenumber of infectious agents that remain after incubation, the greaterthe opsonic activity of the antibody tested. In a cell-mediatedbactericidal assay, opsonic activity is measured by comparing the numberof surviving bacteria between two similar assays, only one of whichcontains the antibody being tested. Alternatively, opsonic activity isdetermined by measuring the number of viable organisms before and afterincubation with a sample antibody. A reduced number of bacteria afterincubation in the presence of antibody indicates a positive opsonizingactivity. In the cell-mediated bactericidal assay, positive opsonizationis determined by culturing the incubation mixture under appropriatebacterial growth conditions. Any reduction in the number of viablebacteria comparing pre-incubation and post-incubation samples, orbetween samples which contain immunoglobulin and those that do not, is apositive reaction.

[0126] Neutrophil-Mediated Opsonophagocytic Bactericidal Assay: Theassay was performed using neutrophils isolated from adult venous bloodby sedimentation using PMN Separation Medium (Robbins Scientific catalogno. 1068-00-0). Forty microliters of antibody, serum, or otherimmunoglobulin source, was added at various dilutions to replicate wellsof a round-bottom microtiter plate. Forty microliters of neutrophils(approximately 2×10⁶ cells per well) was then added to each well,followed immediately by approximately 3×10⁴ mid-log phase bacteria (S.epidermidis strain Hay, ATCC 55133 or S. aureus type 5, gift from S.Wilson, Uniformed Services University of the Health Sciences) in 10 pITryptic Soy Broth (Difco cat. no. 9063-74, or equivalent). Finally, 10μl of immunoglobulin-depleted human serum was added as a source ofactive complement. (Immunoglobulins were removed from human serumcomplement by preincubating the serum with Protein G-agarose and ProteinL-agarose before use in the assay. This depletion of immunoglobulinsminimized the concentrations of anti-staphylococcal antibodies in thecomplement, thereby reducing bacterial killing caused by inherentantibodies in the complement solution.)

[0127] The plates were incubated at 37□C with constant, vigorousshaking. Aliquots of 10 μl were taken from each well at zero time, whenthe sample antibody was first added, and after 2 hours of incubation. Todetermine the number of viable bacteria in each aliquot harvested fromeach sample well, each aliquot was diluted 20-fold in a solution of 0.1%BSA in water (to lyse the PMNs), mixed vigorously by rapid pipetting,and cultured on blood agar plates (Remel, cat. no. 01-202, orequivalent) overnight at 37° C. The opsonic activity was measured bycomparing the number of bacterial colonies observed from the sampletaken at two hours with the number of bacterial colonies observed fromthe sample taken at time zero. Colonies were enumerated using an IPIMinicount Colony Counter.

[0128] Nasal Colonization Assay: The mouse nasal colonization model forS. aureus was based on the work of Kiser et al. (47). Briefly,streptomycin resistant S. aureus type 5 is grown on high salt Columbiaagar (Difco) to promote capsule formation. The bacteria are washed withsterile saline (0.9% NaCl in water) to remove media components andresuspended at ˜10⁸ bacteria/animal dose in saline (0.9% NaCl in water)containing various concentrations and combinations ofanti-staphylococcal or irrelevant control MAbs. Following one hourpreincubation, the bacteria are pelleted and resuspended in a finalvolume of 10 μl per animal dose in either saline or saline containingantibody. Mice that have been maintained on streptomycin-containingwater for 24 hours are sedated with anesthesia. Staphylococci areinjected into the nares of the mice by pipetting without contacting thenose.

[0129] After four to seven days, during which the animals are maintainedon streptomycin-containing water, the animals are sacrificed and thenoses removed surgically and dissected. Nasal tissue is vortexedvigorously in saline (0.9% NaCl in water) plus 0.5% Tween-20 to releaseadherent bacteria and the saline is plated on Columbia blood agar(Remel) and tryptic soy agar (Difco) containing streptomycin todetermine colonization.

[0130] The invention, having been described above, may be betterunderstood by reference to examples. The following examples are intendedfor illustration purposes only, and should not be construed as limitingthe scope of the invention in any way.

EXAMPLE 1 The Production of Hybridomas and Monoclonal Antibodies

[0131] Antibodies were raised against lipoteichoic acid (LTA) from S.aureus by immunizing mice with an LTA conjugate. LTA conjugatesLTA/PspA, LTA/SIA/TT, and LTA/GMBS/TT, prepared as set forth below, wereused.

[0132] To prepare each conjugate, LTA was first derivatized with thiolgroups as follows. S. aureus LTA (Sigma Chemical Co.) was purifiedessentially as described in Fischer et al. (9). The purified LTA wasdiluted to 4 mg/ml with water. One hundred microliters of 0.75 M HEPES,10 mM EDTA, pH 7.5 and 100 μl of 0.1 M SPDP (Pierce) were added to 1 mlof S. aureus LTA. The reaction was incubated for 4 hours at roomtemperature, and then 55 μl of 0.5 M DTT was added and the solution wasdialyzed overnight at 4° C. against 2 mM EDTA, pH 5 (2×1 L). Thereaction resulted in 0.27 mM thiol (LTA-SH) in a 1.2 mL volume, or 0.32μmol thiol, as determined by DTNB assay (2).

[0133] LTA/PspA conjugate was prepared as follows. Three milligrams ofpneumococcal surface protein A (PspA; 188 μl of a 16 mg/ml solution inPBS; prepared essentially as described in Wortham et al. (43) wascombined with 25 μl of 0.75 M HEPES, 10 mM EDTA, pH 7.3 and 17 μl 0.1 MN-hydroxysuccinimidyl iodoacetate (SIA; Bioaffinity Systems) andincubated for 2 hours at room temperature. The volume of the solutionwas then made up to 2 ml with 10 mM sodium acetate, 0.15 M NaCl, 2 mMEDTA, and then concentrated to a final volume of about 150 μl using anUltrafree 4 device (30 kDa cutoff; Amicon). The resulting iodoacetylPspA was then combined with 400 μl of LTA-SH. The pH was raised to 8with 1 M HEPES pH 8, and the reaction proceeded overnight at 4° C.

[0134] The solution was then fractionated on a 1×60 cm S-200HR column,which had been equilibrated with 0.1% deoxycholate (DOC) in PBS. Thevoid volume fractions were pooled, dialyzed into saline (0.15 M NaCl) toremove DOC and PBS, and had an optical density of 0.14 at 280 nm. Theconcentration of protein in the conjugate solution was 0.66 mg/ml by BCAassay (Pierce Chemical Company), and the concentration of phosphate inthe conjugate solution was 0.88 mM by phosphate assay (1).

[0135] LTA/SIA/TT conjugate was prepared as follows. Four milligrams oftetanus toxoid (TT; 280 μl of 14.5 mg/ml; SmithKline Beecham), dilutedto 4 ml with 2 M NaCl was concentrated to 50 μl using an Ultrafree 4centrifugal filter with a 30 kD cutoff (Millipore). The resultingsolution was diluted to 250 μl with 2 M NaCl (TT/2 M NaCl). Seventy-fivemicroliters of 0.25 M HEPES, 2 mM EDTA, pH 7.5 and 8 μlN-hydroxysuccinimide iodoacetate (SIA; Bioaffinity Systems, Roscoe,Ill.) were added to 125 μl of TT/2 M NaCl. The reaction was incubatedfor 2 hours at room temperature and then diluted to about 2 ml with 2 MNaCl. The solution was then concentrated to 150 μl using an Ultrafree 4centrifugal filter.

[0136] One hundred and fifty microliters of the resulting product,iodoacetylated TT, was combined with 400 μl of LTA-SH. The reaction isincubated overnight at 4° C. The reaction was fractionated on a 1×60 cmSephacryl S-200HR column (Pharmacia), equilibrated with 0.1%deoxycholate in PBS. The void volume fractions, containing theLTA/SIA/TT conjugate, were pooled, dialyzed into saline to remove DOCand PBS, and had an optical density of 0.77 at 280 nm. The yield of TTin the conjugate was 0.77 mg/ml by BCA assay (Pierce Chemical Co.). Theconcentration of phosphate in the conjugate solution was 0.77 mM byphosphate assay (1).

[0137] LTA/GMBS/TT conjugate was prepared as follows. Four milligrams ofTT (280 μl of 14.5 mg/ml; SmithKline Beecham), diluted to 4 ml with 2 MNaCl was concentrated to 50 μl using an Ultrafree 4 centrifugal filterwith a 30 kD cutoff (Millipore). The resulting solution was diluted to250 pi with 2 M NaCl (TT/2 M NaCl). Seventy-five microliters of 0.15 MHEPES, 2 mM EDTA, pH 7.5 and 8 μ1 N-hydroxysuccinimide gamma butyricmaleimide (GMBS; Bioaffinity Systems, Roscoe, Ill.) were added to 125 μlof TT/2 M NaCl. The reaction was incubated for 2 hours at roomtemperature and then diluted to 2 mL with 2 M NaCl. The solution wasthen concentrated to 150 μl using an Ultrafree 4 centrifugal filter.

[0138] Four hundred microliters of LTA-SH was added to the concentratedsolution, and the pH was raised to 8 with 1 M HEPES pH 8. The reactionwas incubated overnight at 4° C. The reaction was fractionated on a 1×60cm Sephacryl S-200HR column (Pharmacia), equilibrated with 0.1%deoxycholate in PBS. The void volume fractions, containing theLTA/GMBS/TT conjugate, were pooled and dialyzed into saline to removeDOC and PBS. The yield of TT was 0.83 mg/ml by BCA assay (PierceChemical Co.), and the concentration of phosphate in the conjugatesolution was 1.45 mM by phosphate assay (1).

[0139] The presence of LTA in each of the conjugates was confirmed byWestern blot following 12% SDS-PAGE electrophoresis of the product.

[0140] Twenty-four approximately 4 month old female BALB/c mice wereseparated into six groups and immunized with 10 μg (groups A, C, and E)or 1 μg (groups B, D, and F) of LTA/PspA (groups A and B), LTA/SIA/TT(groups C and D), or LTA/GMBS/TT (groups E and F; Table 1). TABLE 1 LTAImmunization Groups Immun. Group Antigen μg/mouse Mouse Ids A LTA/PspA10 1375-1378 B LTA/PspA 1 1379-1382 C LTA/SIA/TT 10 1383-1386 DLTA/SIA/TT 1 1387-1390 E LTA/GMBS/TT 10 1391-1394 F LTA/GMBS/TT 11395-1398

[0141] All immunizations were administered subcutaneously in 50% RIBIadjuvant. The mice received a boost 21 days after the primaryimmunization, and a second boost 79 days after the primary immunization.Boosts were performed as described for the primary immunizations.Eyebleeds were taken at 0 days, 21 days, 35 days, 79 days, 94 days, and119 days after the primary immunization. Serum collected at 21 days and35 days was tested by ELISA for antibodies to LTA (Table 2). TABLE 2Anti-LTA Titers of Serum Pools prebleed 21 day 35 day Group Antigenμg/mouse titer* titer* titer* A LTA/PSPA 10 77 2885 4748 B LTA/PSPA 1 572668 4667 C LTA/SIA/TT 10 199 51353 54085 D LTA/SIA/TT 1 2520 1152526229 E LTA/GMBS/TT 10 783 11631 140392 F LTA/GMBS/TT 1 10 3635 85832

[0142] Serum collected at 35 days and 79 days was also tested forantibodies to LTA by ELISA, and serum collected at 94 days was tested byELISA, and in an LBE assay against S. epidermidis strain Hay and S.aureus (Table 3). TABLE 3 Comparison of Anti-LTA Titers and LBE TitersLBE** LBE*** ELISA* ELISA* ELISA* S. epi S. aur Group Antigen pg/mouse35 day 79 day 94 day 94 day 94 day A LTA/PSPA 10 16053 4110 65241 531 26B LTA/PSPA 1 23505 8806 156343 150 13 C LTA/SIA/TT 10 153227 39034279505 520 29 D LTA/SIA/TT 1 98798 20135 313256 980 50 E LTA/GMBS/TT 10230410 46859 299995 1903 409 F LTA/GMBS/TT 1 88756 24338 447440 2475 541

[0143] Based on the results of the ELISA assays and LBE assays, day 94and day 119 sera from individual mice in groups E and F were tested byELISA, and in an opsonic assay against S. aureus (Table 4).

[0144] Mouse 1396, which had been immunized with LTA/GMBS/TT, wasselected because serum from the mouse showed a strong signal by anti-LTAELISA, and was opsonic against S. aureus. Mouse 1396 was boosted onemore time at day 134, and then sacrificed on day 141, and spleen removedand used to make hybridomas. TABLE 4 Anti-LTA ELISA and Opsonic Assay ofIndividual Mouse Sera S. aureus opsonic assay** ELISA* day day day dayday Antigen ELISA* day prebleed prebleed 94 94 119 119 119 ID Doseprebleed 119 neat 1:5 neat 1:5 neat 1:5 1:10 1391 10 16364 53 20 68 0 4014 1392 10 18142 47915 19 53 0 1 21 1393 10 18870 65 2 12 37 1394 10 2232126 77 25 78 33 29 50 67 1395 1 29091 7 6 44 16 23 17 39 1396 1 090249 53 20 74 64 1397 1 0 40833 53 0 33 0 1398 1 0 16601 60 27 40 31 37

[0145] Hybridomas were prepared by the general methods of Shulman, Wildeand Kohler; and Bartal and Hirshaut (34, 48). A total of 2.08×10⁸spleenocytes from mouse 1396 were mixed with 2.00×10⁷ SP2/0 mousemyeloma cells (ATCC Catalog number CRL1581) and pelleted bycentrifugation (400×g, 10 minutes at room temperature) and washed inserum free medium. The supernatant was removed to near-dryness andfusion of the cell mixture was accomplished in a sterile 50 mlcentrifuge conical by the addition of 1 ml of warm (37° C.) polyethyleneglycol (PEG; mw 1400; Boehringer Mannheim) over a period of 60-90seconds. The PEG was diluted by slow addition of serum-free medium insuccessive volumes of 1, 2, 4, 8, 16 and 19 mis. The hybridoma cellsuspension was gently resuspended into the medium and the cells pelletedby centrifugation (500×g, 10 minutes at room temperature). Thesupernatant was removed and the cells resuspended in medium RPMI 1640,supplemented with 10% heat-inactivated fetal bovine serum, 0.05 mMhypoxanthine and 16 μM thymidine (HT medium). One hundred pi of thehybridoma cells were planted into 952 wells of 96-well tissue cultureplates. Eight wells (column 1 of plate A) received approximately 2.5×10⁴SP2/0 cells in 100 pi. The SP2/0 cells served as a control for killingby the selection medium added 24 hours later.

[0146] Twenty four hours after preparation of the hybridomas, 100 μl ofRPMI 1640, supplemented with 10% heat-inactivated fetal bovine serums,0.1 mM hypoxanthine, 0.8 μM aminopterin and 32 μM thymidine (HAT medium)was added to each well.

[0147] Forty-eight hours after the preparation of the hybridomas, theSP2/0 cells in plate A, column 1 appeared to be dead, indicating thatthe HAT selection medium had successfully killed the unfused SP2/0cells.

[0148] Ten days after the preparation of the hybridomas, supernatantsfrom all wells were tested by ELISA for the presence of antibodiesreactive with methanol-fixed S. aureus LTA. Based on the results of thispreliminary assay, cells from 12 wells were transferred to a 24-wellculture dish. Three days later, supernatant from these cultures wereretested by ELISA for the presence of antibodies that bind to LTA.

[0149] The absorbance values for eleven of the culture supernatants wereless than 0.100. However, the absorbance value obtained with thesupernatant from hybridoma culture 00-107GG12 was 4.000. This culturewas expanded for further evaluation and cloned into two 96-well culturedishes. Cloning was accomplished by diluting the cell suspension into4.5 viable cells per ml in RPMI 1640, supplemented with 15% fetal bovineserum, 5% Hybridoma SFM (Life Technologies) and 100 pg/ml of kanamycin.

[0150] Ten days later, the supernatants from the hybridoma clones weretested by ELISA for binding to S. aureus LTA. Only one clone, ID12,bound strongly to LTA, with an absorbance of 3.500). In contrast, theabsorbance values for the remaining supernatants were less than 0.220.Hybridoma cloneOO-107GG12 ID12 was expanded and cryopreserved. Isotypedetermination revealed that both the original hybridoma (00-107GG12) andits clone (00-107GG12 ID12) were mouse IgG_(2a) heavy chains with kappalight chains. The monoclonal antibody produced by hybridoma 00-107GG12ID12 was designated M120.

EXAMPLE 2 Opsonic Activity of M120

[0151] Opsonic assays were carried out substantially as described aboveunder the heading “Neutrophil-mediated Opsonophagocytic BacteriacidalAssay”. M120 was purified from ascites essentially as described by themanufacturer of MEP Hypercel gel (BiSepra). Thirty-three ml of buffer A(50 mM Tris, 5 mM EDTA, pH 8) was added to 17 ml of mouse ascites, andthen centrifuged for 15 minutes at 4000 rpm in an Eppendorf model 581 ORcentrifuge using rotor A462. The solution was filtered using WhatmanGD/XP PES 0.45μ membrane (cat. no. 6994-2504) and the volume of dilutedascites was 47 ml after filtering. The solution was loaded onto a 1 cm×7cm MEP hypercel column that had been equilibrated with buffer A, at arate of 1.8 ml/min. The column was washed with buffer A, and then withbuffer A+25 mM sodium caprylate until the OD₂₈₀ was <0.05. The columnwas then washed with water until the OD₂₈₀<0.05. The column was elutedwith buffer B (50 mM sodium acetate, 5 mM EDTA, pH 4) and the eluentcollected at a rate of 70 drops/min. The main peak (pool A) and itstrailing end (pool B) were pooled separately and dialyzed against PBS(2×2L) at 4° C. The dialyzed solution was sterile filtered using aMillex GV device (Millipore). By OD₂₈₀, pool A contained 3.2 mg/mlantibody, and pool B contained 0.25 mg/ml antibody.

[0152] First, the opsonic activity of M120 was determined against S.aureus Type 5 (Table 5). TABLE 5 Opsonic Activity of M120 (200 μg/ml)against S. aureus Type 5 Description Assay 1 Assay 2 Assay 3 Assay 4PMNs alone 0 0 0 0 C' alone 0 9 0 0 PMNs + C 0 16 25 11 M120 alone 20 00 0 M120 + PMNs + C' 73 84 85 85

[0153] Next, the opsonic activity of M120 was determined against S.epidermidis strain Hay (Table 6). This assay was also performed asdescribed above under the heading “Neutrophil-Mediated OpsonophagocyticBactericidal Assay”. TABLE 6 Opsonic activity of M120 against S.epidermidis strain Hay MAb Description (μg/ml) % killed PMNs alone 0 C'alone 0 PMNs + C' 0 M120 alone 200 17 M120 + PMNs + C' 200 95 ″ 100 97 ″30 75 ″ 10 87 ″ 3.3 55

[0154] A similar assay was used to determine the opsonic activity ofMAb-391.4 against S. epidermidis strain Hay (Table 7). Thus, MAb-391.4,which was raised against UV-killed S. aureus, has strong opsonicactivity against S. epidermidis strain Hay. TABLE 7 Opsonic activity ofMAb-391.4 against S. epidermidis strain Hay MAb Description (μg/ml) %killed PMNs alone 10.9 C' alone 0 PMNs + C' 0 M120 alone 120 24.7 M120 +PMNs + C' 120 81.9 ″ 50 57.9

EXAMPLE 3 Cloning of the M120 Variable Regions

[0155] Total RNA was isolated from 4×10⁶ frozen 00-107 GG12 ID12hybridoma cells using the Midi RNA Isolation kit (Qiagen) following themanufacturer's procedure. The RNA was dissolved in 10 mM Tris, 0.1 mMEDTA (pH 8.4) containing 0.03U/μg Prime RNase Inhibitor (Sigma) to afinal concentration of 0.25 μg/μl.

[0156]FIG. 3 shows the strategy for cloning the variable region genes.The total RNA (2 μg) was converted to cDNA by using Superscript II-MMLVReverse Transcriptase (Life Technologies) and mouse Kappa chain-specificprimer (JSBX-18; SEQ ID NO: 5) and a mouse heavy chain-specific primer(JSBX-25A; SEQ ID NO: 6) according to the manufacturer's procedures (seeFIG. 4 for primer sequences). The first strand cDNA synthesis productswere then purified using a Centricon-30 concentrator device (Amicon). Ofthe 40 μl of cDNA recovered, 5 μl was used as template DNA for PCR.Typical PCR amplification reactions (50 μl) contained template DNA, 30pmoles of the appropriate primers (JSBX-9A, 11 A, and 18 for lightchains, SEQ ID NOs: 3-5; JSBX-1, 4 and JSBX-25A for heavy chains, SEQ IDNOs: 1, 2, and 6), 2.5 units of ExTaq polymerase (PanVera), 1×ExTaqreaction buffer, 200 μM dNTP, 2 mM MgCl₂. The template was denatured byan initial incubation at 96° C. for 3 min. The products were amplifiedby 30 thermal cycles of 96° C. for 1 min., 60° C. for 30 sec., 72° C.for 30 seconds. The PCR products from the successful reactions werepurified using the Nucleospin PCR Purification system (Clontech) as permanufacturer's procedure.

[0157] The PCR products (approximately 400 base pairs each) were thencloned into a bacterial vector, pGEM T (Promega) for DNA sequencedetermination. PCR fragments were ligated into pGEM T, a T/A stylecloning vector, following the manufacturer's procedures using a 3:1insert to vector molar ratio. One half (5 μl) of the ligation reactionswere used to transform Ultracompetent XL1 Blue cells (Stratagene) as perthe manufacturer's procedure. Bacterial clones containing plasmids withDNA inserts were identified using diagnostic restriction enzymedigestions with DraIII and BsiWI (for heavy chain clones) or DraIII andEcoRV (for light chain clones) (New England Biolabs). The DNA sequencesof plasmids containing inserts of the appropriate size (˜400 bp) werethen determined. The plasmid containing the A120 heavy chain sequencewas designated pJSB16-6 and the plasmid containing the A120 light chainvariable region was designated pJSB17-23. The final consensus DNAsequences of the light chain and heavy chain variable regions are shownin FIG. 5 and FIG. 6, respectively.

[0158] Having sequenced the variable regions of both M110 and M120, wecompared them. The homology was striking at both the DNA and amino acidlevels. As set forth in FIG. 9 there is a 96% homology at the DNA level,with 662 out of 687 bases the same Further, at the amino acid level,there is a 94% homology, with 216 amino acids out of 225 the same, asset forth in FIG. 10. As noted above, M120 was raised agains S. aureusLTA, while Ml 10 was raised against S. epidermis strain Hay. Bothantibodies exhibit opsonic activity against both S. epidermis and S.aureus. The high level of homology between the M110 and M120 variableregions may suggest a common structural motif that contributes to theopsonic capability of the antibodies.

EXAMPLE 4 Production of Recombinant Chimeric Mouse/Human AntibodyMolecules

[0159] The heavy and light chain variable regions were then subclonedinto a mammalian expression plasmid vector for production of recombinantchimeric mouse/human antibody molecules. The human/mouse chimera of theM120 antibody is designated A120, and the human/mouse chimera of theM110 antibody is designated A110 (See U.S. patent application Ser. No.09/097,055, filed Jun. 15, 1998).

[0160] As set forth below, vectors were designed that expressrecombinant antibody molecules under the control of CMV transcriptionalpromoters. The chimeric heavy chains are expressed as a fusion of aheavy chain variable region and a human IgG1 constant domain. Thechimeric light chains are expressed as a fusion of a light chainvariable region and a human kappa chain constant region. The chimericlight chain cDNA contains a mouse kappa intron between the variableregion and the human kappa constant region. After splicing, the variableregion becomes fused to a human Kappa constant region exon. Theselectable marker for the vector in mammalian cells is Neomycinresistance (resistance to G418).

[0161] The variable region gene fragments of M120 were re-amplified byPCR using primers that adapted the fragments for cloning into theexpression vector (see FIG. 4, JSBX-46 through JSBX-49, SEQ ID NOs: 7-9and 18). The heavy chain front primer (JSBX-46; SEQ ID NO: 7) includes a5′ tail that encodes the C-terminus of the heavy chain leader and aBSiWI restriction site for cloning, while the heavy chain reverse primer(JSBX-47; SEQ ID NO: 8) adds a 3′ EcoRI restriction site for cloning.This results in the addition of two amino acids, glutamine (E) andphenylalanine (F) between the heavy chain variable region and the humanIgG1 constant region. The light chain front primer (JSBX-48; SEQ ID NO:9) introduces a 5′ tail that encodes the two C-terminal amino acids ofthe light chain leader and an Age/restriction site for cloning purposes.The light chain reverse primer (JSBX-49; SEQ ID NO: 18) adds a 3′ DNAsequence for the joining region-Kappa exon splice junction followed by aBstBI restriction site for cloning. The variable regions werere-amplified from the plasmid DNA using vector pJSB16-6 for the heavychain variable region and vector pJSB17-23 for the light chain variableregion. PCR reactions were performed as described above. Following a 3minute incubation at 96° C., the PCR parameters were 30 thermal cyclesof 58° C. for 30 seconds, 70° C. for 30 seconds, and 96° C. for 1minute.

[0162] The heavy chain variable region PCR product was digested withBsiWI and EcoRI (New England Biolabs), purified using a Nucleospin PCRPurification column (Clontech), as described by the manufacturer, andligated into BsiWI/EcoRI/PfIMI-digested and gel-purified pJRS383 vectorusing the Takara Ligation Kit (Panvera) following the manufacturer'sprocedure. The ligation mix was then transformed into XL1 Blue cells(Stratagene), resulting in plasmid mammalian expression vector pJSB23-1(FIG. 7). The light chain variable region PCR product (approximately 350bp) was digested with AgeI and BstBI (New England Biolabs), and purifiedusing a Nucleospin PCR Purification column (Clontech) as described bythe manufacturer. The light chain variable region fragment was ligatedinto pJRS384 that had been AgeI/BstBI/XcmI-digested and gel-purifiedusing the Takara Ligation Kit (Panvera) following the manufacturer'sprocedure. The ligation mix was transformed into XLI Blue cells(Stratagene), resulting in mammalian expression plasmid pJSB24 (FIG. 8).

[0163] Because of the similarity between the A110 and the A120 antibodysequences, we decided to construct mammalian cell expression plasmidsthat contained the A120 heavy and light chain variable regions in abi-cistronic plasmid, as well as plasmids that combined the A110 heavychain variable region with the A120 light chain variable region, and theA110 light chain variable region with the A120 heavy chain variableregion, in order to investigate the binding and opsonic properties ofthe different antibodies. Construction of the bi-cistronic vectors wasdone in a step-wise fashion, in which the heavy and light chain variableregions of A120 were cloned into a bi-cistonic expression plasmidalready containing the A110 light and heavy chain variable regions(pJRS354, FIG. 11), replacing the A110 light chain variable region,heavy chain variable region, or both. The plasmid pJRS354 was digestedwith ClaI and XhoI (New England Biolabs), the digestion products wereseparated on an agarose gel and the backbone fragment was cut out andgel purified using a Nucleospin Gel Fragment DNA Purification column(Clontech), as described by the manufacturer. The plasmid pJSB24 wasdigested with ClaI and XhoI (New England Biolabs), the digestionproducts were separated on an agarose gel and the light chain variableregion fragment was cut out and gel purified using a Nucleospin GelFragment DNA Purification column (Clontech), as described by themanufacturer. These fragments were then ligated together using theTakara Ligation Kit (Panvera) following the manufacturer's procedure.The resulting bi-cistronic expression vector, pJSB25-3 (FIG. 12), whichcontained the A120 antibody light chain variable region and the A110antibody heavy chain variable region, was then used for antibodyproduction in transfected mammalian cells after sequence confirmation ofthe variable regions.

[0164] The two other bi-cistronic plasmids were constructed in a similarmanner. The plasmid pJSB25-3 was digested with BspEI and NotI (NewEngland Biolabs), the digestion products were separated on an agarosegel and the backbone fragment was cut out and gel purified using aNucleospin Gel Fragment DNA Purification column (Clontech), as describedby the manufacturer. The plasmid pJSB23-1 was digested with BspEl andNofl (New England Biolabs),), the digestion products were separated onan agarose gel and the heavy chain variable region fragment was cut outand gel purified using a Nucleospin Gel Fragment DNA Purification column(Clontech), as described by the manufacturer. These fragments wereligated together using the Takara Ligation Kit (Panvera) following themanufacturer's procedure. The resulting bi-cistronic expression vector,pJSB26 (FIG. 13), which contained the light and heavy chain variableregions of the A120 antibody, was then used for antibody production intransfected mammalian cells.

[0165] The plasmid pJRS354 was digested with BspEI and NotI (New EnglandBiolabs), the digestion products were separated on an agarose gel andthe backbone fragment was cut out and gel purified using a NucleospinGel Fragment DNA Purification column (Clontech), as described by themanufacturer. The plasmid pJSB23-1 was digested with BspEI and NotI (NewEngland Biolabs), the digestion products were separated on an agarosegel and the heavy chain variable region fragment was cut out and gelpurified using a Nucleospin Gel Fragment DNA Purification column(Clontech), as described by the manufacturer. These fragments wereligated together using the Takara Ligation Kit (Panvera) following themanufacturer's procedure and transformed into XL1Blue cells(Stratagene). The resulting bi-cistronic expression vector, pJSB27 (FIG.14), which contained the heavy chain variable region of the A120antibody and the light chain variable region of the A110 antibody, wasthen used for antibody production in transfected mammalian cells.

EXAMPLE 5 Comparison of the A120 and A110 anti-LTA Human/Mouse ChimericAntibodies

[0166] Anti-LTA human/mouse chimeric antibody A110 was previouslydescribed in U.S. patent application Ser. No. 09/097,055, which isherein incorporated by reference. The binding activities of anti-LTAhuman/mouse chimeric antibodies A110 and A120 were compared in an ELISAassays against LTA.

[0167] Dilutions of A120 supernatant were compared to dilutions ofpurified A110 antibody in an immunoassay as described above under theheading “Binding Assays”, subheading “Immunoassay on LTA”. Briefly, thewells of a 96-well plate were coated with 1 μg/ml of S. aureus LTA forthree hours at room temperature. After washing, dilutions of purifiedA110 antibody or A120 supernatant in PBS-T were added to quadruplicatewells and incubated for 30 to 60 minutes at room temperature. Afterwashing, HRP-conjugated gamma-specific goat anti-human IgG, diluted1:5000, was added to each well and incubated for 30 to 60 minutes atroom temperature. After removing the secondary antibody and washing, 100μl TMB substrate was added to each well and incubated for 15 minutes atroom temperature. One hundred microliters of TMB stop reagent was thenadded to each well to stop the reaction, and the absorbance of each wellat 450 nm was determined. The results of the anti-LTA ELISA assay areshown in Table 8. TABLE 8 Binding of A110 and A120 to LTA-coated platesA120 A110 supernatant (ng/ml) A₄₅₀ dilution A₄₅₀ 40 2.691 10 4.000 201.741 20 3.967 10 0.952 40 3.927 5 0.555 80 3.327 2.5 0.322 160 2.8241.25 0.180 320 1.907 0.625 0.115 640 1.148 PBS-T 0.050 PBS-T 0.052

[0168] This assay shows that monoclonal antibody A120, like A110, bindsto LTA of S. aureus. In order to compare the binding affinity of the twoantibodies, A120 was purified using Protein G Ultralink (Pierce) per themanufacturer's procedure, and the two antibodies were tested for bindingto LTA in a second ELISA assay.

[0169] Dilutions of purified A110 and A120 antibodies were compared forbinding to LTA in an ELISA assay, using substantially the same protocolas above. The data for the anti-LTA ELISA using dilutions of purifiedantibodies are shown in Table 9. TABLE 9 Binding of purified A110 andA120 to LTA-coated plates antibody concentration A₄₅₀ A₄₅₀ (ng/ml) A110A120 8000 3.921 3.566 2000 3.922 3.078 500 3.960 1.445 125 3.838 0.42231.25 2.398 0.131 7.8125 0.903 0.068 1.953 0.276 0.054 PBS-T 0.047 0.048

[0170] These data demonstrate that A110 has a greater affinity for S.aureus LTA than does A120. This difference is particularly striking atan antibody concentration of 125 ng/ml, where A120 gives an ELISA signalof 0.422, and A110 gives a signal that is nearly ten times stronger.

EXAMPLE 6 Comparison of the Opsonic Activity of Anti-LTA Antibodies A110and A120

[0171] Purified human/mouse chimeric A110 and A120, and mouse M120 MAbswere assayed for their opsonic activity as described above, under theheading “Neutrophil-Mediated Opsonophagocytic Bactericidal Assay.”Briefly, dilutions of purified A110, A120, or M120 antibodies werecombined with neutrophils (PMNs) in the wells of a microtiter plate.Mid-log phase bacteria were added to each well, followed byimmunoglobulin-depleted human serum, which serves as a source ofcomplement (C′). Samples were incubated for 2 hours at 37° C., and werethen plated on blood agar and incubated overnight to determine thenumber of live bacteria remaining. The opsonic activity is expressed as“% killed”, which is determined according to the following formula: %killed=100%−N_(2hr)/N_(0hr), where N_(2hr) is the number of coloniesformed after a 2 hour incubation with antibody, PMNs, and C′, andN_(0hr) is the number of colonies formed after a 0 hour incubation.Control reactions lacked one of the above components. Table 10 shows theresults of the opsonic activity assay for antibodies A110, A120, andM120. TABLE 10 Opsonic activity of A110, A120, and M120 against S.epidermidis strain Hay MAb conc. Antibody Description (μg/ml) % killedA110 purified human/mouse 100 99 chimeric ″ anti-LTA MAb 50 96 ″ ″ 10 96A120 purified human/mouse 100 100 chimeric ″ anti-LTA MAb 50 99 ″ ″ 1094 M120 purified mouse anti-LTA 100 99 MAb ″ ″ 50 97 ″ ″ 10 94 PMNsalone ″ N/A 0 C' alone ″ N/A 0 PMNs + C' (no MAb) ″ N/A 14 A110 alone ″100 0 A120 alone ″ 100 0 M120 alone ″ 100 8

[0172] These data demonstrate that MAbs A110, A120, and M120 are equallyactive in the opsonic activity assay described herein. Thus, thechimerization of M120 to make A120 has little or no effect on theopsonic activity of the antibody, and the two different anti-LTAchimeric antibodies, A110 and A120, are of comparable activity.

EXAMPLE 7 Transient Production of Recombinant Chimeric Mouse/Human A120Antibodies

[0173] The plasmids pJSB25, pJSB26 and pJSB27 were transected into COScells grown in IMDM plus 10% fetal bovine serum, using Superfect(Qiagen) in 6 well tissue culture wells as described by themanufacturer. After two days the supernatant was assayed for theproduction of chimeric antibody and for the capability for the expressedantibody to bind to S. aureus LTA antigen.

[0174] Antibody production assays were preformed in 8-well strips from96-well microtiter plates (Maxisorp F8; Nunc, Inc.) coated at a 1:500dilution with a goat antihuman Fc (Pierce). The plates are covered withpressure sensitive film and incubated overnight at 4° C. Plates werethen washed once with Wash solution (Imidazole/NaCl/0.4%Tween-20). Onehundred microliters of culture supernatant dilutions were then appliedto duplicate wells and allowed to incubate for 60 minutes on a platerotator at room temperature. The plates were washed seven times withWash solution. A Goat anti-Human IgG H+L-HRP (Zymed) conjugate wasdiluted 1:4000 in the sample/conjugate diluent. One hundred microliterswas added to the samples, and then incubated on a plate rotator for 60minutes at room temperature. The samples were washed as above and thenincubated with 100 μL/well of TMB developing substrate (BioFx) for 1minute at room temperature. The binding reaction was stopped with 100μL/well of Quench buffer (BioFx) and the absorbance value at 450 nm wasdetermined using an automated microtiter plate ELISA reader. This assay(see FIG. 15) demonstrates that the transfection of cells with thisplasmid construct results in the cells producing a molecule containingboth human IgG and Kappa domains.

[0175] The supernatants were then assayed for the ability of theexpressed antibodies to bind to lipoteichoic acid. The activity assayswere preformed in 8-well strips from 96-well microtiter plates (MaxisorpF8; Nunc, Inc.) coated at 1 μg/mL with S. aureus LTA (Sigma) using PBS.The plates were covered and incubated overnight at 4° C. Plates werethen washed once with PBS. One hundred microliters of culturesupernatant dilutions were then applied to duplicate wells and allowedto incubate for 60 minutes on a plate rotator at room temperature. Theplates were washed seven times with Wash solution. Goat anti-Human IgGH+L-HRP (Zymed) was diluted 1:4000 in the sample/conjugate diluent, and100 μl were added to the samples, and then incubated on a plate rotatorfor 60 minutes at room temperature. The samples were washed as above andthen incubated with 100 μL/well of TMB developing substrate (BioFx) for10-15 minutes on a plate rotator at room temperature. The bindingreaction was stopped with 100 μL/well of Quench buffer (BioFx) and theabsorbance value at 450 nm was determined using an automated microtiterplate ELISA reader. As a positive control, the original human/mousechimeric antibody A110 (produced by plasmid pJRS354) was used. Thisassay (FIG. 16) demonstrates that the transfection of cells with theseplasmid constructs results in the cells producing a molecule that bindsto the S. aureus LTA antigen.

[0176] These data demonstrate that the chimeric human antibody directedagainst LTA is opsonic and enhances survival against staphylococci. Inaddition, the antibody promotes clearance of the staphylococci from theblood. Thus antibody to LTA provides prophylactic and therapeuticcapabilities against staphylococcal infections and vaccines using LTA orpeptide mimeotopes of LTA that induce anti-LTA antibodies would alsohave prophylactic capabilities.

EXAMPLE 8 Human Antibodies that Bind LTA

[0177] Rather than humanizing a mouse antibody to minimize the HAMAresponse during treatment as described above, a skilled artisan canisolate a protective anti-LTA antibody that is fully human. There are anumber of well-known alternative strategies one of ordinary skill in theart may use to produce completely human recombinant antibodies. One isthe generation of antibodies using phage display technologies (59, 63).Specifically, human RNA is used to produce a cDNA library of antibodyheavy and light chain fragments expressed on the surface ofbacteriophage. These libraries can be used to probe against the antigenof interest (i.e., LTA) and the phage that bind, because of the antibodyexpressed on the surface, are then isolated. The DNA encoding thevariable regions is sequenced and cloned for antibody expression.

[0178] Another method of producing human antibodies employs “humanized”mice. These transgenic mice have had their own antibody genes replacedwith a portion of the human antibody gene complex so that uponinoculation with antigen, they produce human antibodies (57, 59, 60, 61,63). The antibody producing cells that result can then be incorporatedinto the standard hybridoma technology for the establishment of specificmonoclonal antibody producing cell lines.

[0179] Recombinant human antibodies are also produced by isolatingantibody-producing B cells from human volunteers that have a robustanti-LTA response. Using fluorescence activated cell sorting (FACS) andfluorescently labeled LTA, cells producing the anti-LTA antibodies canbe separated from the other cells. The RNA can then be extracted and thesequence of the reactive antibody variable regions determined (58, 62).The DNA sequence of the functional variable regions can be synthesizedor cloned into mammalian expression vectors for large-scale humanrecombinant antibody production.

CONCLUSION

[0180] Monoclonal antibodies were raised in mice against S. aureus LTA.One hybridoma that produced antibodies that bound strongly to LTA in anELISA assay was subcloned further. Hybridoma subclone 00-107GG12 ID12produced an IgG₂a monoclonal antibody with a kappa light chain thatbound strongly to LTA. The antibody produced by this hybridoma wasdesignated M120 (Example 1).

[0181] M120 was tested in an opsonophagocytic bacteriocidal assay foropsonic activity against S. aureus type 5 and S. epidermidis strain Hay.The antibody was mixed with PMNs and complement, which was derived fromhuman serum that had been depleted of anti-S. aureus and anti-S.epidermidis antibodies, and then tested for activity against thebacteria. M120 showed opsonic activity against both S. aureus and S.epidermidis, killing 95% of S. epidermidis and an average of 82% of S.aureus at 200 μg/ml (Example 2, Tables 5 and 6). MAb-391.4, which wasraised to UV-killed S. aureus, was tested for opsonic activity againstS. epidermidis strain Hay in a similar assay, and showed 81.9% killing(Table 7).

[0182] The M120 variable regions were then cloned and sequenced, and thesequence compared to another anti-LTA antibody, M110. Surprisingly, M110and M120 were found to share about 94% sequence identity at the aminoacid level, and about 96% sequence identity at the nucleotide level. Athird anti-LTA antibody, MAb-391.4, was also sequenced compared to theother two. The three antibodies share 88% sequence identity at the aminoacid level. This high level of sequence identity may suggest that theantibodies bind to a common epitope on LTA (Example 3, FIGS. 9 and 10).Human/mouse chimeric antibodies were then made, fusing the heavy chainvariable region of either M120 or M110 to a human IgG1 constant region,and the light chain variable region of either M120 or M110 to a humankappa light chain constant region. The human/mouse chimera of M120 isreferred to as A120 and the human/mouse chimera of M110 is referred toas A110. Because of the similarity between the two antibodies, anantibody that contained the heavy chain of A110 and the light chain ofA120, designated A120a, was made. Similarly, an antibody that containedthe light chain of A110 and the heavy chain of A120, designated A120b,was also made (Example 4).

[0183] The human/mouse chimeric antibodies A120 and A110 were tested fortheir ability to bind to LTA in an ELISA assay. Both chimeric antibodiesbound strongly to LTA, indicating that replacing the mouse constantregions with human constant regions had little effect on the bindingproperties of the antibodies (Example 5, Tables 8 and 9). Next, theopsonic activity of chimeric antibodies A110 and A120, and of M120, werecompared in an opsonic assay against S. epidermidis strain Hay. Allthree antibodies showed at least 94% killing of S. epidermidis. Theseresults show that the chimeric antibodies are strongly opsonic againstS. epidermidis, and because they have a reduced HAMA response in humans,they should be suitable therapeutic molecules for fighting Gram-positivebacterial infections (Example 6, Table 10).

[0184] Finally, three of the chimeric antibodies, A120, A120a, and A120bwere produced in COS cells and tested for the ability to bind to S.aureus LTA. All three chimeric antibodies bound to LTA in the ELISAassay, with A120 and A120a showing the strongest binding. These resultssuggest that M110 and M120 do bind to a similar or overlapping epitopeon LTA, because antibodies that have variable regions from both retainthe ability to bind to the antigen. These results may indicate that aparticular epitope on LTA is able to elicit antibodies that are opsonicagainst S. aureus and S. epidermidis. This epitope may be moreaccessible than others, or may be positioned such that antibodies thatare bound are ideally situated to attract the factors required foropsonization of the bacterium.

[0185] Previously, it was unclear whether a monoclonal antibody couldenhance phagocytosis, because the polyclonal sera that were usedcontained many different antibodies that bound to many differentepitopes on the surface of the bacteria, and the sum of this collectivebinding and activities may have accounted for the overall activity ofthe serum. Here, we demonstrate that monoclonal antibodies, which bindto a single epitope on the surface of bacteria, can be opsonic againstthat bacteria. We have also demonstrated that monoclonal antibodiesraised against LTA can have that activity, and that those antibodies maybe opsonic for a number of different types of Gram-positive bacteria.

[0186] Furthermore, we have shown that three different monoclonalantibodies, one of which was raised to whole S. epidermidis, one topurified and conjugated LTA from S. aureus, and one to whole UV-killedS. aureus, share a striking degree of homology. This level of homologybetween monoclonal antibodies that were raised to similar antigens indifferent mice has previously not been shown. In fact, it has long beenbelieved that antibodies have evolved the ability to bind identicalantigens using very dissimilar determinants to provide the body with avery broad antibody repertoire. The level of homology between the M110,M120, and MAb-391.4 variable regions may indicate that opsonicantibodies to LTA recognize a nearly identical epitope using nearlyidentical modes of binding, and that this mode of binding is importantto their functional activity. Furthermore, the epitope to which theantibodies bind appears to be highly conserved between S. epidermidisand S. aureus, and may be common to most, if not all, Gram-positivebacteria. Monoclonal antibodies to this epitope may, therefore, bebroadly opsonic against a wide range of bacteria, allowing researchersto develop a few antibodies that will have broad opsonic and protectiveactivity against many Gram-positive bacteria.

[0187] The following literature references are herein specificallyincorporated by reference:

[0188] 1. Ames, B. N. 1966. Assay of inorganic phosphate, totalphosphate and phosphatase, Methods in Enzymology 8:115-118.

[0189] 2. ElIman, G. L. 1959. Tissue Sulfhydryl Groups, Arch. Biochem. &Biophys. 82: 70

[0190] 3. Endl, J.; Seidl, H. P.; Fiedler, F.; and Schleifer, K. H.1983. Chemical composition and structure of cell wall teichoic acid ofstaphylococci, Arch Microbiol, 135: 215-223.

[0191] 4. Espersen, F.; Hertz, J. B.; and Hoiby, N. 1981.Cross-reactions between Staphylococcus epidermis and 23 other bacterialspecies, Acta Path. Microbial. Scand., Sect. B. 89: 253-260.

[0192] 5. Exley A. R.; Cohen J.; Buurman W.; Owen R.; Hanson G.; LumleyJ.; Aulakh J. M.; Bodmer M.; Riddell A.; Stephens S.; et al. 1990.Monoclonal antibody to TNF in severe septic shock, Lancet 335:1275-1277.

[0193] 6. Fattom A.; Shepherd S.; Karakawa W. 1992. Capsularpolysaccharide serotyping scheme for Staphylococcus epidermidis, J.Clin. Micro. 30: 3270-3273.

[0194] 7. Fischer, Gerald W. Broadly reactive opsonic antibodies thatreact with common staphylococcal antigens, U.S. Pat. No. 5,571,511,issued Nov. 5, 1996.

[0195] 8. Fischer, Gerald W. Directed human immune globulin for theprevention and treatment of staphylococcal infections, U.S. Pat. No.5,955,074, issued Sep. 21, 1999.

[0196] 9. Fischer W.; Koch H. U.; Haas R. 1983. Improved preparation oflipoteichoic acids, Eur. J. Biochem. 133: 523-530.

[0197] 10. Fleer, A.; Senders R. C.; Visser M. R.; Bijlmer R. P.;Gerards L. J.; Kraaijeveld C. A.; Verhoef J. 1983. Septicemia due tocoagulase-negative staphylococci in a neonatal intensive care unit:clinical and bacteriological features and contaminated parenteral fluidsas a source of sepsis, Pediatr. Infect. Dis. 2: 426-431.

[0198] 11. Fournier, Jean-Michel. 1991. Staphylococcus Aureus, Vaccinesand Immunotherapy, Ch. 13, pp.166-171.

[0199] 12. Garrett, Laurie. 1994. The Revenge of the Germs or Just KeepInventing New Drugs, The Coming Plague, Ch. 13, Farrar, Straus andGiroux, NY, (ed.), pp. 411-456.

[0200] 13. Genarro, A. (ed.) 1990. Remington's Pharmaceutical Sciences,18^(th) Edition, Mack Publishing, Easton, Pa.

[0201] 14. Hancock, I. C. 1997. Bacterial cell surface carbohydrates:Structure and assembly, Biochem. Soc. Trans. 25:183-187.

[0202] 15. Jendeberg, Lena; Nilsson, Peter; Larsson, Antonella; Denker,Per; Uhlen, Mathias; Nilsson, Bjorn; Nygren, Per-Ake. 1997. Engineeringof Fc1 and Fc3 from human immunoglobulin G to analyse subclassspecificity for Staphylococcal Protein A, J. Immunol. Methods 201:25-34.

[0203] 16. Kojima Y.; Tojo M.; Goldmann D. A.; Tosteson T. D.; Pier G.B. 1990. Antibody to the capsular polysaccharide/adhesin protectsrabbits against catheter-related bacteremia due to coagulase-negativestaphylococci, J. Infect. Dis. 162: 435-441.

[0204] 17. Krieger, Monty; Joiner, Keith A. Method for treatingGram-positive septicemia, U.S. Pat. No. 5,624,904, issued Apr. 29, 1997.

[0205] 18. Lee, J. C. 1996. The prospects for developing a vaccineagainst Staphylococcus aureus, Trends in Micro. 4: 162-66.

[0206] 19. LoBuglio A. F.; Wheeler R. H.; Trang J.; Haynes A.; RogersK.; Harvey E. B.; Sun L.; Ghrayeb J.; Khazaeli M. B. 1989. Mouse/humanchimeric monoclonal antibody in man: kinetics and immune response,P.N.A.S. 86: 4220-4224.

[0207] 20. Nakamura, K. et al. 1999. Uptake and release of budesonidefrom mucoadhesive, pH-sensitive copolymers and their application tonasal delivery. J. Control. Release 61:329-335.

[0208] 21. Natsume, H., S. lwata, K. Ohtak, M. Miyamoto, M. Yamaguchi,K. Hosoya, and D. Kobayashi. 1999. Screening of cationic compounds as anabsorption enhancer for nasal drug delivery. Int. J. Pharma. 185:1-12.

[0209] 22. Naumova, I. B.; Kuznetsov, V. D.; Kudrina, K. S.; andBezzubenkova, A. P. 1980. The Occurrence of Teichoic Acids inStreptomycetes, Arch. Microbiol. 126: 71-75.

[0210] 23. Navarre, William Wiley and Schneewind, Olaf. 1999. Surfaceproteins of Gram-positive bacteria and mechanisms of their targeting tothe cell wall envelope, Microbiology and Molecular Biology Reviews63:174-229.

[0211] 24. Osland, Arve; Grov, Arne; and Oeding, Per. 1980.Immunochemical analysis of the teichoic acid from Staphylococcussimulans, Acta Path. Microbiol. Scand., Sect. B., 88:121-123.

[0212] 25. Patrick, C. C. 1990. Coagulase-negative staphylococci:Pathogens with increasing clinical Significance, J. Pediatr. 116:497-507.

[0213] 26. Peterson, Phillip K.; Verhoef, Jan; Sabath, L. D.; and Quie,Paul G. 1977. Effect of Protein A on staphylococcal opsonization,Infection and Immunity 15: 760-764.

[0214] 27. Peterson, Phillip K.; Wilkinson, Brian J.; Kim, Youngki;Schmeling, David; and Quie, Paul G. 1978. Influence of Encapsulation onStaphylococcal Opsonization and Phagocytosis by Human PolymorphonuclearLeukocytes, Infection and Immunity 19: 943-949.

[0215] 28. Quie, Paul G.; Hill, Harry R.; and Davis, Todd A. 1974.Defective phagocytosis of Staphylococci, Annals New York Academy ofSciences, pp.233-243.

[0216] 29. Ramkissoon-Ganorkar, C. et al. 1999. Modulatinginsulin-release profile from pH/thermosensivite polymeric beads throughpolymer molecular weight. J. Contr. Release 59:287-298.

[0217] 30. Raynor, Robert H.; Scott, David F.; and Best, Gary K. 1981.Lipoteichoic acid inhibition of phagocytosis of Staphylococcus aureus byHuman Polymorphonuclear Leukocytes, Clinical Immunology andImmunopathology 19: 181-189.

[0218] 31. Romero-Vivas J.; Rubio M.; Fernandez C.; Picazo J. J. 1995.Mortality associated with nosocomial bacteremia due tomethicillin-resistant Staphylococcus aureus, Clin. Infect. Dis. 21:1417-23.

[0219] 32. Salton, M. R. J. 1994. The Bacterial Cell Envelope—AHistorical Perspective, in J.-M. Ghuyson and R. Hakenbeck (ed.),Bacterial Cell Wall, Elsevier Science BV, Amsterdam, pp. 1-22.

[0220] 33. Schwab, U. E., A. E. Wold, J. L. Carson, M. W. Leigh, P.-W.Cheng, P. H. Gilligan and T. F. Boat. 1993. Increased adherence ofStaphylococcus aureus from cystic fibrosis lungs to airway epithelialcells. Am. Rev. Respir. Dis. 148:365-369.

[0221] 34. Shulman, M.; Wilde, C. D.; Kohler, G. 1978. A Better CellLine for Making Hybridomas Secreting Specific Antibodies, Nature 276:269-270.

[0222] 35. Soto, N., A. Vaghjimal, A. Stahl-Avicolli, J. Protic, L.Lutwick and E. Chapnick. 1999. Bacitracin versus mupirocin forStaphylococcus aureus nasal colonization. Infect. Cont. Hosp. Epidem.20:351-353.

[0223] 36. Suzuki, Y. and Y. Makino. 1999. Mucosal drug delivery usingcellulose derivative as a functional polymer. J. Control. Release.62:101-107.

[0224] 37. Takada H.; Kawabata Y.; Arakaki R.; Kusumoto S.; Fukase K.;Suda Y.; Yoshimura T.; Kokeguchi S.; Kato K.; Komuro T.; et al. 1995.Molecular and structural requirements of a lipoteichoic acid fromEnterococcus hirae ATCC 9790 for cytokine-inducing, antitumor, andantigenic activities, Infection and Immunity 63: 57-65.

[0225] 38. Takeda S.; Pier G. B.; Kojima Y.; Tojo M.; Muller E.;Tosteson T.; Goldmann D. A. 1991. Protection against endocarditis due toStaphylococcus epidermidis by immunization with capsularpolysaccharide/adhesin, Circulation 86: 2539-2546.

[0226] 39. Timmerman C. P.; Besnier J. M.; De Graaf L.; Torensma R.;Verkley A. J.; Fleer A.; Verhoef J. 1991. Characterisation andfunctional aspects of monoclonal antibodies specific for surfaceproteins of coagulase-negative staphylococci, J. Med. Micro. 35: 65-71.

[0227] 40. Tomasz, Alexander. 2000. The Staphylococcal Cell Wall, in V.A. Fischetti et al. (ed.) Gram-Positive Pathogens, Ch. 36, pp. 351-355.

[0228] 41. Waldvogel, Francis A. 1990. Staphylococcus Aureus (IncludingToxic Shock Syndrome), in Mandell, G. L. et al. (ed.) Principles andPractices of Infectious Diseases, Third Edition, Churchill Livingstone,N.Y., Ch. 173, pp. 1489-1510.

[0229] 42. West, Timothy E.; Cantey, J. R.; Apicella, Michael A.; andBurdash, N. M. 1983. Detection of anti-teichoic acid immunoglobulin Gantibodies in experimental Staphylococcus epidermidis endocarditis,Infection and Immunity 42:1020-1026.

[0230] 43. Wortham, Charles; Grinberg, Luba; Kaslow, David C.; Briles,David E.; McDaniel, Larry S.; Lees, Andrew; Flora, Michael; Snapper,Clifford M.; and Mond, James J. 1998. Enhanced protective antibodyresponse to PspA after intranasal or subcutaneous injections of PspAgenetically fused to Granulocyte-Macrophage Colony-Stimulating Factor orInterleukin-2, Infection and Immunity 66: 1513-1520.

[0231] 44. Sambrook, Joseph; Russell, David W. 1989. Molecular Cloning:A Laboratoiy Manual, 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y.

[0232] 45. Ausubel et al. (ed.) 1989. Current Protocols in MolecularBiology, John Wiley & Sons.

[0233] 46. Merkus, F. W., J. C. Verhoef, N. G. Schipper, and E. Marttin.1999. Cyclodextrins in nasal drug delivery. Advan. Drug Deliv. Rev.36:41-57.

[0234] 47. Kiser, Kevin B.; Cantey-Kiser, Jean M.; Lee, Jean C. 1999.Development and characterization of a Staphylococcus aureus nasalcolonization model in mice. Infection and Immunity 67: 5001-5006.

[0235] 48. Bartal, Arie H.; Hirshaut, Yashar. 1987. Current Methods inHybridoma Formation Bartal, A. H. et al. (ed.) Methods of HybridomaFormation, Humana Press, Clifton, N.J.

[0236] 49. De Kimpe, S. J., M. Kengatharan, C. Thiemermann, J. R. Vane.1995. The cell wall components peptidoglycan and lipoteichoic acid fromS. aureus act in synergy to cause shock and multiple organ failure.Proc. Nat Acad. Sci. (USA) 92:10359-10363.

[0237] 50. Carruthers, M. M., W. J. Kabat. 1983. Mediation ofstaphylococcal adherence to mucosal cells by lipoteichoic acid. InfectImmun. 40: 444-6.

[0238] 51. Chugh T D, Burns G J, Shuhaiber H J, Bahr G M. 1990.Adherence of Staphylococcus epidermidis to fibrin-platelet clots invitro mediated by lipoteichoic acid. Infect Immun. 58: 315-9.

[0239] 52. Granato D, Perotti F, Masserey I, Rouvet M, Golliard M,Servin A, Brassart D. 1999. Cell surface-associated lipoteichoic acidacts as an adhesion factor for attachment of Lactobacillus johnsonii Lalto human enterocyte-like Caco-2 cells. Appl Environ Microbiol.65:1071-7.

[0240] 53. Nealon T J, Mattingly S J. 1984. Role of cellularlipoteichoic acids in mediating adherence of serotype III strains ofgroup B streptococci to human embryonic, fetal, and adult epithelialcells. Infect Immun. 43: 523-30.

[0241] 54. Teti G, Tomasello F, Chiofalo M S, Orefici G, Mastroeni P.1987. Adherence of group B streptococci to adult and neonatal epithelialcells mediated by lipoteichoic acid. Infect Immun. 55: 3057-64.

[0242] 55. Nickerson, K. G.; Tao, M.-H.; Chen, H.-T.; Larrick, J.;Kabat, E. A. 1995. Human and mouse monoclonal antibodies to blood groupA substance, which are nearly identical immunochemically, use radicallydifferent primary sequences. J. Biol. Chem. 270: 12457-12465.

[0243] 56. Fleury, D.; Daniels, R. S.; Skehel, J. J.; Knossow, M.;Bizebard, T. 2000. Structural evidence for recognition of a singleepitope by two distinct antibodies. Proteins 40: 572-578.

[0244] 57. Green, L. L., M. C. Hardy, et al. (1994). “Antigen-specifichuman monoclonal antibodies from mice engineered with human Ig heavy andlight chain YACs.” Nat Genet 7(1): 13-21.

[0245] 58. Kantor, A. B., C. E. Merrill, et al. (1995). “Development ofthe antibody repertoire as revealed by single-cell PCR of FACS-sortedB-cell subsets.” Ann N Y Acad Sci 764: 224-7.

[0246] 59. Low, N. M., P. H. Holliger, et al. (1996). “Mimicking somatichypermutation: affinity maturation of antibodies displayed onbacteriophage using a bacterial mutator strain.” J Mol Biol 260(3):359-68.

[0247] 60. Wagner, S. D., A. V. Popov, et al. (1994). “The diversity ofantigen-specific monoclonal antibodies from transgenic mice bearinghuman immunoglobulin gene miniloci.” Eur J Immunol 24(11): 2672-81.

[0248] 61. Wagner, S. D., G. T. Williams, et al. (1994). “Antibodiesgenerated from human immunoglobulin miniloci in transgenic mice.”Nucleic Acids Res 22(8): 1389-93.

[0249] 62. Wang, X. and B. D. Stollar (2000). “Human immunoglobulinvariable region gene analysis by single cell RT-PCR.” J Immunol Methods244(1-2): 217-25.

[0250] 63. Winter, G., A. D. Griffiths, et al. (1994). “Makingantibodies by phage display technology.” Annu Rev Immunol 12: 433-55.

[0251] 64. Borrebaeck, Carl A. K. 1995. Antibody Engineering, 2^(nd)Ed., Oxford University Press, NY.

[0252] 65. Harlow, Ed; Lane, David. 1988. Antibodies: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0253] Having now fully described the invention, it will be appreciatedby those skilled in the art that the invention can be performed within arange of equivalents and conditions without departing from the spiritand scope of the invention and without undue experimentation. Inaddition, while the invention has been described in light of certainembodiments and examples, the inventors believe that it is capable offurther modifications. This application is intended to cover anyvariations, uses, or adaptations of the invention which follow thegeneral principles set forth above.

[0254] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

What is claimed is:
 1. A MAb comprising at least one light chain and atleast one heavy chain, wherein said at least one light chain comprises apolypeptide comprising an amino acid sequence having at least 70%identity with a light chain variable region selected from Seq. ID Nos.16, 10, and 21; wherein said at least one heavy chain comprises apolypeptide comprising an amino acid sequence having at least 70%identity with a heavy chain variable region selected from Seq. ID Nos.12, 17, or 22; and wherein said MAb specifically binds to LTA.
 2. TheMab according to claim 1, wherein the percents identity are at least80%.
 3. The Mab according to claim 1, wherein the percents identity areat least 90%.
 4. The MAb of claim 1, comprising at least one variableregion having an amino acid sequence selected from Seq. ID Nos. 10, 12,16, 17, 21, and
 22. 5. The MAb according to claim 1, wherein at leastone light chain, at least one heavy chain, or both are chimeric orhumanized.
 6. The MAb according to claim 1, wherein at least one lightchain, at least one heavy chain, or both, are human.
 7. The MAbaccording to claim 1, comprising a heavy chain constant region; whereinsaid constant region comprises human IgG, IgA, IgM, or IgD sequence. 8.The MAb of claim 1, comprising a Fab, Fab′, F(ab′)₂, Fv, SFv, scFv.
 9. Apolypeptide comprising an amino acid sequence having at least 70%identity with a light chain variable region selected from Seq. ID Nos.16, 10, and 21; wherein said polypeptide is capable of functioning as avariable region, or portion thereof, in a MAb that specifically binds toLTA.
 10. The polypeptide according to claim 9, comprising at least oneregion having at least 88% identity with a sequence selected from aminoacids 24-33, 49-55, and 88-73 of Seq. ID Nos. 10, 16, or 21; whereinsaid region is capable of functioning as a CDR, or portion thereof, in aMAb that specifically binds to LTA.
 11. The polypeptide according toclaim 9, comprising at least one region having at least 82% identitywith a sequence selected from amino acids amino acids 1-23, 34-38,56-87, and 97-106 of Seq. ID Nos. 10, 16, or 21; wherein said region iscapable of functioning as a framework region, or portion thereof, in aMAb that specifically binds to LTA.
 12. A MAb light chain comprising thepolypeptide according to claim
 9. 13. The Mab light chain according toclaim 12, wherein said light chain is chimeric, humanized, or human. 14.The MAb light chain according to claim 12, comprising a light chainconstant region comprising human kappa or lambda sequence.
 15. Apolypeptide comprising an amino acid sequence having at least 70%identity with a heavy chain variable region selected from Seq. ID Nos.12, 17, or 22; wherein said polypeptide is capable of functioning as avariable region, or portion thereof, in a MAb that specifically binds toLTA.
 16. The polypeptide according to claim 15, comprising at least oneregion having at least 80% identity with a sequence selected from aminoacids 26-35, and 50-69 of Seq. ID Nos. 12, 17, or 22; wherein saidregion is capable of functioning as a CDR, or portion thereof, in a MAbthat specifically binds to LTA.
 17. The polypeptide according to claim15, comprising at least one region having at least 80% identity with asequence selected from amino acids amino acids 1-25, 36-49, 70-101, and115-125 Seq. ID Nos. 12, 17, or 22; wherein said region is capable offunctioning as a framework region, or portion thereof, in a MAb thatspecifically binds to LTA.
 18. A MAb heavy chain comprising thepolypeptide according to claim
 15. 19. The Mab heavy chain according toclaim 18, wherein said heavy chain is chimeric, humanized, or human. 20.The MAb heavy chain according to claim 18, comprising a heavy chainconstant region comprising human IgG, IgA, IgM, or IgD sequence.
 21. AMAb comprising at least one light chain and at least one heavy chain,wherein said MAb specifically binds LTA; and wherein said at least onelight chain comprises a variable region having at least one CDRcomprising a sequence selected from amino acids 24-33, 49-55, or 88-73of Seq. ID Nos. 10, 16, or 21; or wherein said at least one light chaincomprises a variable region having at least one CDR comprising asequence selected from amino acids 1-25, 36-49, 70-101, or 115-125 ofSeq. ID Nos. 12, 17, or
 22. 22. A Mab according to claim 21, comprisingat least one variable domain selected from A110, A110b, A120, A120b, and391.4.
 23. A hybridoma cell line expressing a MAb according to claim 22.24. A pharmaceutical composition comprising one or more Mabs accordingto claim 1 and a pharmaceutically acceptable carrier.
 25. Thepharmaceutical composition according to claim 24, wherein saidcomposition is opsonic for S. epidermidis and S. aureus.
 26. Thepharmaceutical composition of claim 25, further comprising at least oneantibody that binds to peptidoglycan (PepG) of Gram-positive bacteria.27. A method of treating a patient comprising administering thepharmaceutical composition of claim
 24. 28. The method of claim 27,wherein the composition is administered internasally.
 29. The method ofclaim 27, wherein the pharmaceutical composition further comprises atleast one antibody that binds to peptidoglycan (PepG) of Gram-positivebacteria.
 30. The method of claim 29, wherein the composition isadministered internasally.
 31. A method of making the MAb of claim 1comprising the steps of: a) selecting at least one Mab that specificallybinds to at least of LTA, or a peptide mimeotope of LTA that inducesanti-LTA antibodies; b) determining the polypeptide sequence of thelight chain variable region of said at least one Mab; c) selecting apolypeptide sequence having at least 70% identity with a light chainvariable region selected from Seq. ID Nos. 16, 10, and 21; d)determining the polypeptide sequence of the heavy chain variable regionof said at least one Mab; e) selecting a polypeptide sequence having atleast 70% identity with a heavy chain variable region selected from Seq.ID Nos. 12, 17, or 22; f) combining a light chain comprising apolypeptide sequence of step c) with a heavy chain comprising apolypeptide sequence of step e).
 32. A method of making the polypeptideof claim 9, comprising the steps of: a) selecting at least one Mab thatspecifically binds to at least of LTA, or a peptide mimeotope of LTAthat induces anti-LTA antibodies; b) determining the polypeptidesequence of the light chain variable region of said at least one Mab; d)selecting a polypeptide sequence having at least 70% identity with alight chain variable region selected from Seq. ID Nos. 16, 10, and 21.33. A method of making the polypeptide of claim 15, comprising the stepsof: a) selecting at least one Mab that specifically binds to at least ofLTA, or a peptide mimeotope of LTA that induces anti-LTA antibodies; b)determining the polypeptide sequence of the heavy chain variable regionof said at least one Mab; d) selecting a polypeptide sequence having atleast 70% identity with a heavy chain variable region selected from Seq.ID Nos. 12, 17, or
 22. 34. A purified nucleic acid encoding thepolypeptide of claim
 9. 35. A purified nucleic acid encoding thepolypeptide of claim
 15. 36. A production system comprising, 1) a cell;and 2) one or more recombinant nucleic acids capable of directing theexpression of a Mab according to claim
 1. 37. A method of identifyinghighly antigenic and highly conserved epitopes comprising the steps of:a) selecting a multiplicity of MAbs that specifically binds to animmunogen; b) determining the polypeptide sequence of the variableregions of said MAbs; d) identifying regions of identity in thepolypeptide sequence of at least two of said Mabs, said regions ofidentity comprising at least one of 1) at least 70% identity of lightchain variable regions, at least 70% identity of heavy chain variableregions, at least 70% identity over 3 complementarity determiningregions (CDRs) in a variable region, at least 75% identity over at leasttwo CDRs in a variable region; at least 80% identity in a CDR; and atleast 70% identity in the framework regions (FRs) of a variable region.38. A collection of Mabs that bind to LTA comprising, a multiplicity ofMabs according to claim
 1. 39. The collection of claim 38, wherein thecollection comprises one or more of M110, M120, 391.4, or a chimeric orhumanized derivative thereof.